Chiral phosphoramidite auxiliaries and methods of their use

ABSTRACT

Disclosed are P-stereogenic groups that may be used in the synthesis of compounds including stereochemically enriched P-stereogenic phosphorothioates. P-stereogenic groups may be provided in nucleoside phosphoramidites including a sugar bonded to a nucleobase and to a stereochemically enriched phosphoramidite as well as methods of their use and methods of making them.

FIELD OF THE INVENTION

The invention relates to chiral auxiliaries and reagents useful fordiastereoselective syntheses of P-stereogenic phosphites, phosphates,and phosphorothioates. The invention also relates to the preparation ofoligonucleotides and methods of making chiral reagents.

BACKGROUND

Oligonucleotides including phosphorothioate phosphodiesters have twopossible oligonucleotide diastereomers for each P-stereogenicphosphorothioate. Many oligonucleotide therapeutics include multipleP-stereogenic phosphorothioates and thus have 2^(n) possiblediastereomers, where n is the number of P-stereogenic phosphorothioates.For example, an oligonucleotide containing six phosphorothioatephosphodiesters has 64 possible different diastereomers, whichcollectively can form over 10¹⁹ different diastereomeric mixtures.Separation of oligonucleotide diastereomers is impractical, in view ofthe material losses in the form of other diastereomers and thecomplexity of method development for oligonucleotide diastereomerseparation. Accordingly, synthesis of oligonucleotides includingP-stereogenic phosphorothioates requires development of reagents andmethods for stereoselective introduction of P-stereogenicphosphorothioates. The currently available chiral reagents typicallyrequire lengthy synthetic routes. New reagents and methods for thesynthesis of oligonucleotides including stereochemically enrichedP-stereogenic phosphorothioates are needed.

SUMMARY OF THE INVENTION

In general, the present invention provides P-stereogenic groups,compounds containing them, and methods for diastereoselective synthesisof, e.g., oligonucleotides including stereochemically enrichedinternucleoside phosphorothioates.

In one aspect, the invention provides a P-stereogenic group of formula(IA), (IB), (IC), or (ID):

-   -   where    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring; and    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.

In some embodiments, the P-stereogenic group is of formula (IA) or (IB).In certain embodiments, R¹ and R², together with the atoms to which eachis attached, combine to form an optionally substituted 5- to 8-memberedring (e.g., an optionally substituted 5- to 8-membered carbocyclic ring(e.g., optionally substituted 5- to 8-membered ring is an optionallysubstituted 6-membered carbocyclic ring)).

In particular embodiments, the P-stereogenic group is of the followingstructure:

In further embodiments, R³ is H. In yet further embodiments, R⁴ is H. Instill further embodiments, R³ and R⁴ are each H.

In certain embodiments, the P-stereogenic group is of formula (IIA),(IIB), (IIA′), or (IIB′).

In another aspect, the invention provides a compound of formula (IIIA),(IIIB), (IIIC), or (IIID):

-   -   where    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   A is an optionally substituted C₁₋₁₂ alkyl, optionally        substituted C₃₋₁₀ cycloalkyl, optionally substituted C₃₋₁₀        cycloalkyl-C₁₋₆-alkyl, optionally substituted C₁₋₉ heterocyclyl,        optionally substituted C₁₋₉ heterocyclyl-C₁₋₆-alkyl, sugar        analogue, nucleoside, nucleotide, or oligonucleotide;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring; and    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.

In some embodiments, the compound is of formula (IIIA) or (IIIB).

In certain embodiments, R¹ and R², together with the atoms to which eachis attached, combine to form an optionally substituted 5- to 8-memberedring (e.g., optionally substituted 5- to 8-membered carbocyclic ring(e.g., optionally substituted 6-membered carbocyclic ring)).

In particular embodiments, the compound is of the following structure:

In further embodiments, R³ is H. In yet further embodiments, R⁴ is H. Instill further embodiments, R³ and R⁴ are each H.

In other embodiments, the compound is of formula (IVA), (IVB), (IVA′),or (IVB′).

In yet another aspect, the invention provides a nucleosidephosphoramidite including a sugar bonded to a nucleobase and to aphosphoramidite of the following structure:

-   -   where    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring; and    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.

In some embodiments, the nucleoside phosphoramidite includes aphosphoramidite of formula (VA) or (VB).

In certain embodiments, the nucleoside phosphoramidite is of thefollowing structure:

-   -   where    -   B¹ is a nucleobase;    -   Y¹ is H or C₁₋₆ alkyl;    -   R⁵ is H, O-protected hydroxyl, optionally substituted C₁₋₆        alkoxy, or halogen; and    -   R⁶ is a hydroxyl protecting group.

In particular embodiments, the nucleoside phosphoramidite is of formula(VIA) or (VIB).

In further embodiments, R⁵ is hydrogen, halogen, or optionallysubstituted C₁₋₆ alkoxy. In yet further embodiments, R⁵ is hydrogen,fluoro, or methoxy. In still further embodiments, R⁶ is dimethoxytrityl.

In some embodiments, R¹ and R², together with the atoms to which each isattached, combine to form an optionally substituted 5- to 8-memberedring (e.g., optionally substituted 5- to 8-membered carbocyclic ring(e.g., optionally substituted 6-membered carbocyclic ring)).

In certain embodiments, the phosphoramidite is of the followingstructure:

In further embodiments, R³ is H. In yet further embodiments, R⁴ is H. Instill further embodiments, R³ and R⁴ are each H.

In still another aspect, the invention provides a compound of formula:

-   -   where    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   X is a halogen or pseudohalogen;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring; and    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.

In certain embodiments, the compound is of formula (VIIIA) or (VIIIB).

In some embodiments, R¹ and R², together with the atoms to which each isattached, combine to form an optionally substituted 5- to 8-memberedring (e.g., optionally substituted 5- to 8-member carbocyclic ring(e.g., optionally substituted 6-member carbocyclic ring)).

In particular embodiments, the compound is of the following structure:

In some embodiments, the compound is of formula (IXA), (IXB), (IXA′), or(IXB′).

In further embodiments, R³ is H. In yet further embodiments, R⁴ is H. Instill further embodiments, R³ and R⁴ are each H.

In a further aspect, the invention provides a method of preparing acomposition containing an oligonucleotide including a stereochemicallyenriched internucleoside phosphorothioate by (i) reacting the nucleosidephosphoramidite disclosed herein with a coupling activator and anucleoside including a 5′-hydroxyl or an oligonucleotide including a5′-hydroxyl, (ii) reacting with an electrophilic source of acyl, and(iii) reacting with a sulfurizing agent to produce the oligonucleotidecontaining a stereochemically enriched internucleoside phosphorothioatetriester.

In some embodiments, the method further includes converting thephosphorothioate triester into a phosphorothioate diester by reactingthe phosphorothioate triester with an aqueous base.

In particular embodiments, the coupling activator is5-(benzylthio)-1H-tetrazole (BTT), N-(phenyl)imidazoliumtrifluoromethanesulfonate (PhIMT), or N-(cyanomethyl)pyrrolidiniumtrifluoromethanesulfonate (CMPT). In certain embodiments, the couplingactivator is CMPT.

In further embodiments, the nucleoside is a 2′-deoxyribonucleoside. Inyet further embodiments, the electrophilic source of acyl is an acidanhydride (e.g., acetic anhydride or trifluoroacetic anhydride). Instill further embodiments, the sulfurizing agent is3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione(DDTT).

In yet further aspect, the invention provides a method of preparing thenucleoside phosphoramidite including a sugar bonded to a nucleobase andphosphoramidite of the following structure:

-   -   where    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring; and    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl;    -   by reacting a sugar bonded to a nucleobase with a compound of        formula (VIIIA), (VIIIB), (VIIIC), or (VIIID):

-   -   where    -   X is a halogen or pseudohalogen.

In particular embodiments, the nucleoside phosphoramidite is of formula(VA) or (VB), and a sugar bonded to a nucleobase is reacted with acompound of formula (VIIIA) or (VIIIB).

In a further aspect, the invention provides an oligonucleotide (e.g., anoligonucleotide having a total of 2-100 nucleosides (e.g., 2 to 50 or 2to 35) including one or more (e.g., 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to2, or 1) internucleoside groups independently selected from the groupconsisting of linkers of formula (XIA) and (XIB):

-   -   where    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring;    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl; and    -   R⁷ is acyl (e.g., alkanoyl).

In certain embodiments, R¹ and R², together with the atoms to which eachis attached, combine to form an optionally substituted 5- to 8-memberedring (e.g., optionally substituted 5- to 8-membered carbocyclic ring(e.g., optionally substituted 6-membered carbocyclic ring)).

In some embodiments, the one or more (e.g., 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, or 1) internucleoside groups are selected from the group oflinkers of formula (XIIIA), (XIIIB), (XIIIA′), and (XIIIB′):

where

-   -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl; and    -   R⁷ is acyl (e.g., alkanoyl).

In further embodiments, R³ is H. In yet further embodiments, R⁴ is H. Instill further embodiments, R³ and R⁴ are each H.

In a yet further aspect, the invention provides an oligonucleotide(e.g., an oligonucleotide having a total of 2-100 nucleosides (e.g., 2to 50 or 2 to 35) including one or more (e.g., 1 to 6, 1 to 5, 1 to 4, 1to 3, 1 to 2, or 1) internucleoside groups independently selected fromthe group consisting of linkers of formula (XIIA) and (XIIB):

where

-   -   is a single carbon-carbon bond or a double carbon-carbon bond;

each of R¹ and R² is independently an optionally substituted C₁₋₆ alkylor optionally substituted C₆₋₁₀ aryl, or R¹ and R², together with theatoms to which each is attached, combine to form an optionallysubstituted 5- to 8-membered ring;

-   -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl; and    -   R⁷ is acyl (e.g., alkanoyl).

In certain embodiments, R¹ and R², together with the atoms to which eachis attached, combine to form an optionally substituted 5- to 8-memberedring (e.g., optionally substituted 5- to 8-membered carbocyclic ring(e.g., optionally substituted 6-membered carbocyclic ring)).

In some embodiments, the one or more (e.g., 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, or 1) internucleoside groups are selected from the group oflinkers of formula (XIVA), (XIVB), (XIVA′), and (XIVB′):

where

-   -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl; and    -   R⁷ is acyl (e.g., alkanoyl).

In further embodiments, R³ is H. In yet further embodiments, R⁴ is H. Instill further embodiments, R³ and R⁴ are each H.

Definitions

The term “abasic spacer,” as used herein, refers to internucleoside,abasic spacers known in the art, e.g., those described in WO2018/035380. For example, an abasic spacer may be a group of formula(X′):

-   -   where    -   each of R^(A1) and R^(A2) is independently H, —OR^(A4), or        —N(R^(A4))(R^(A5)); where R^(A4) is optionally substituted C₁₋₁₆        alkyl, optionally substituted C₂₋₁₆ heteroalkyl, or a protecting        group, and R^(A5) is H optionally substituted C₁₋₁₆ alkyl,        optionally substituted C₂₋₁₆ heteroalkyl, or a protecting group;        and    -   each of m1, m2, m3, and m4 is independently an integer from 0 to        11, provided that the quaternary carbon in the structure above        is bonded to 0 or 1 atoms other than carbon and hydrogen, and        provided that the sum of m1, m2, m3 and m4 is 11 or less.

The term “about,” as used herein, represents a value that is ±10% of therecited value.

The term “acyl,” as used herein, represents a group of formula —C(O)—R¹,where R¹ is H, alkyl, aryl, or heteroaryl. Acyl may be optionallysubstituted as defined for the group present as R¹ in acyl. Acyl, inwhich R¹ is alkyl (e.g., optionally substituted alkyl), may be referredto as an alkanoyl. Acyl, in which R¹ is aryl (e.g., optionallysubstituted aryl), may be referred to as an aryloyl. Acyl, in which R¹is heteroaryl (e.g., optionally substituted heteroaryl), may be referredto as an heteroaryloyl.

The term “acyloxy,” as used herein, represents a group of formula —OR,where R is acyl. Acyloxy may be optionally substituted as defined foracyl. Acyloxy, in which R is alkanoyl (e.g., optionally substitutedalkanoyl), may be referred to as an alkanoyloxy. Acyl, in which R isaryloyl (e.g., optionally substituted aryloyl), may be referred to as anaryloyloxy. Acyl, in which R is heteroaryloyl (e.g., optionallysubstituted heteroaryloyl), may be referred to as an heteroaryloyloxy.

The term “alkanoylamino,” as used herein, represents a group of formula—NHR, where R is alkanoyl.

The term “alkenyl,” as used herein, represents acyclic monovalentstraight or branched chain hydrocarbon groups of containing one, two, orthree carbon-carbon double bonds. An unsubstituted alkenyl includes 2 to16 carbon atoms. Non-limiting examples of the alkenyl groups includeethenyl, prop-1-enyl, prop-2-enyl, 1-methylethenyl, but-1-enyl,but-2-enyl, but-3-enyl, 1-methylprop-1-enyl, 2-methylprop-1-enyl, and1-methylprop-2-enyl. Alkenyl groups may be optionally substituted with1, 2, 3, or 4 substituent groups selected, independently, from the groupconsisting of aryl, cycloalkyl, heterocyclyl (e.g., heteroaryl), asdefined herein, and the substituent groups described for alkyl.

The term “alkenylene,” as used herein, refers to a straight or branchedchain alkenyl group with one hydrogen removed, thereby rendering thisgroup divalent. The valency of alkenylene defined herein does notinclude the optional substituents. Non-limiting examples of thealkenylene groups include ethen-1,1-diyl; ethen-1,2-diyl;prop-1-en-1,1-diyl, prop-2-en-1,1-diyl; prop-1-en-1,2-diyl,prop-1-en-1,3-diyl; prop-2-en-1,1-diyl; prop-2-en-1,2-diyl;but-1-en-1,1-diyl; but-1-en-1,2-diyl; but-1-en-1,3-diyl;but-1-en-1,4-diyl; but-2-en-1,1-diyl; but-2-en-1,2-diyl;but-2-en-1,3-diyl; but-2-en-1,4-diyl; but-2-en-2,3-diyl;but-3-en-1,1-diyl; but-3-en-1,2-diyl; but-3-en-1,3-diyl;but-3-en-2,3-diyl; buta-1,2-dien-1,1-diyl; buta-1,2-dien-1,3-diyl;buta-1,2-dien-1,4-diyl; buta-1,3-dien-1,1-diyl; buta-1,3-dien-1,2-diyl;buta-1,3-dien-1,3-diyl; buta-1,3-dien-1,4-diyl; buta-1,3-dien-2,3-diyl;buta-2,3-dien-1,1-diyl; and buta-2,3-dien-1,2-diyl. The alkenylene groupmay be unsubstituted or substituted (e.g., optionally substitutedalkenylene) as described for alkenyl groups.

The term “alkenoxy,” as used herein, represents a chemical substituentof formula —OR, where R is an alkenyl group, unless otherwise specified.An alkenyloxy group may be substituted or unsubstituted (e.g.,optionally substituted alkenyloxy) as described herein for alkyl groups.

The term “alkoxy,” as used herein, represents a chemical substituent offormula —OR, where R is a C₁₋₆ alkyl group, unless otherwise specified.In some embodiments, the alkyl group can be optionally substituted inthe manner described for alkyl groups.

The term “alkoxycarbonyl,” as used herein, represents a chemicalsubstituent of formula —COOR, where R is alkyl. An alkoxycarbonyl groupmay be substituted or unsubstituted (e.g., optionally substitutedalkoxycarbonyl) as described herein for alkyl groups.

The term “alkyl,” as used herein, refers to an acyclic straight orbranched chain saturated hydrocarbon group having from 1 to 16 carbons(when unsubstituted), unless otherwise specified. Alkyl groups areexemplified by methyl; ethyl; n- and iso-propyl; n-, sec-, iso- andtert-butyl; neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) alkoxy; (2) alkylsulfinyl; (3) amino; (4) arylalkoxy; (5)(arylalkyl)aza; (6) azido; (7) halo; (8) (heterocyclyl)oxy; (9)(heterocyclyl)aza; (10) hydroxy; (11) nitro; (12) oxo; (13) aryloxy;(14) sulfide; (15) thioalkoxy; (16) thiol; (17) aryl; (18) —CO₂R^(A),where R^(A) is selected from the group consisting of (a) alkyl, (b)aryl, (c) hydrogen, and (d) arylalkyl; (19) —C(O)NR^(B)R^(C), where eachof R^(B) and R^(C) is, independently, selected from the group consistingof (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl; (20) —SO₂R^(D),where R^(D) is selected from the group consisting of (a) alkyl, (b)aryl, and (c) arylalkyl; (21) —SO₂NR^(E)R^(F), where each of R^(E) andR^(F) is, independently, selected from the group consisting of (a)hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl; (22) silyl; (23) cyano;and (24) —S(O)R^(H) where R^(H) is selected from the group consisting of(a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl. In someembodiments, each of these groups can be further substituted withunsubstituted substituents as described herein for each respectivegroup.

The term “alkylamino,” as used herein, refers to a group —N(R^(N1))₂, inwhich each R^(N1) is independently H or alkyl, provided that at leastone R^(N1) is alkyl. Alkylamino may be optionally substituted; eachalkyl in optionally substituted alkylamino is independently andoptionally substituted as described for alkyl.

The term “alkylaminocarbonyl,” as used herein, represents a chemicalsubstituent of formula R—(CO)—, where R is alkylamino.

The term “alkylaminoalkylaminocarbonyl,” as used herein, represents achemical substituent of formula R¹—R²—NH—(CO)—, where R¹ is alkylamino,and R² is alkylene.

The term “alkylene,” as used herein, refers to a saturated divalenthydrocarbon group derived from a straight or branched chain saturatedhydrocarbon by the removal of two hydrogen atoms. The valency ofalkylene defined herein does not include the optional substituents.Non-limiting examples of the alkylene group include methylene,ethane-1,2-diyl, ethane-1,1-diyl, propane-1,3-diyl, propane-1,2-diyl,propane-1,1-diyl, propane-2,2-diyl, butane-1,4-diyl, butane-1,3-diyl,butane-1,2-diyl, butane-1,1-diyl, and butane-2,2-diyl, butane-2,3-diyl.The term “C_(x-y) alkylene” represents alkylene groups having between xand y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and 6, andexemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Insome embodiments, the alkylene can be optionally substituted with 1, 2,3, or 4 substituent groups as defined herein for an alkyl group.Similarly, the suffix “ene” designates a divalent radical of thecorresponding monovalent radical as defined herein. For example,alkenylene, alkynylene, arylene, aryl alkylene, cycloalkylene,cycloalkyl alkylene, cycloalkenylene, heteroarylene, heteroarylalkylene, heterocyclylene, and heterocyclyl alkylene are divalent formsof alkenyl, alkynyl, aryl, aryl alkyl, cycloalkyl, cycloalkyl alkylcycloalkenyl, heteroaryl, heteroaryl alkyl, heterocyclyl, andheterocyclyl alkyl. For aryl alkylene, cycloalkyl alkylene, heteroarylalkylene, and heterocyclyl alkylene, the two valences in the group maybe located in the acyclic portion only or one in the cyclic portion andone in the acyclic portion. For example, the alkylene group of anaryl-C₁-alkylene or a heterocyclyl-C₁-alkylene can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “alkyleneoxy,” as used herein, refers to a divalent group—R—O—, in which R is alkylene. Alkylene in alkyleneoxy may beunsubstituted or substituted (e.g., optionally substituted alkyleneoxy)as described for alkyl.

The term “alkylsulfonyl,” as used herein, refers to a group —SO₂—R,where R is alkyl.

The term “alkylsulfonyloxy,” as used herein, refers to a group —OR,where R is alkylsulfonyl.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain hydrocarbon groups of from two to sixteen carbon atomscontaining at least one carbon-carbon triple bond and is exemplified byethynyl, 1-propynyl, and the like. Alkynyl groups may be optionallysubstituted with 1, 2, 3, or 4 substituent groups that are selected,independently, from aryl, alkenyl, cycloalkyl, and heterocyclyl (e.g.,heteroaryl), as described herein, and the substituent groups describedfor alkyl.

The term “alkynylene,” as used herein, refers to a straight-chain orbranched-chain divalent substituent including one or two carbon-carbontriple bonds and containing only C and H when unsubstituted. Anunsubstituted alkynylene contains from two to sixteen carbon atoms,unless otherwise specified. The valency of alkynylene defined hereindoes not include the optional substituents. Non-limiting examples of thealkenylene groups include ethyn-1,2-diyl; prop-1-yn-1,3-diyl;prop-2-yn-1,1-diyl; but-1-yn-1,3-diyl; but-1-yn-1,4-diyl;but-2-yn-1,1-diyl; but-2-yn-1,4-diyl; but-3-yn-1,1-diyl;but-3-yn-1,2-diyl; but-3-yn-2,2-diyl; and buta-1,3-diyn-1,4-diyl. Thealkynylene group may be unsubstituted or substituted (e.g., optionallysubstituted alkynylene) as described for alkynyl groups.

The term “amino,” as used herein, represents —N(R^(N1))₂, where, ifamino is unsubstituted, both R^(N1) are H; or, if amino is substituted,each R^(N1) is independently H, —OH, —NO₂, —N(R^(N2))₂, —N(R)SO₂OR^(N2),—SO₂R^(N2), —SOR^(N2), —COOR^(N2), an N-protecting group, alkyl,alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl,cycloalkenyl, heteroalkyl, or heterocyclyl, provided that at least oneR^(N1) is not H, and where each R^(N2) is independently H, alkyl, oraryl. Each of the substituents may itself be unsubstituted orsubstituted with unsubstituted substituent(s) defined herein for eachrespective group. In some embodiments, amino is unsubstituted amino(i.e., —NH₂) or substituted amino (e.g., —NHR^(N1)), where R^(N1) isindependently —OH, —SO₂OR^(N2), —SO₂R^(N2), —SOR^(N2), —COOR^(N2),optionally substituted alkyl, or optionally substituted aryl, and eachR^(N2) can be optionally substituted alkyl or optionally substitutedaryl. In some embodiments, substituted amino may be alkylamino, in whichthe alkyl group is optionally substituted as described herein for alkyl.In further embodiments, substituted amino may be dialkylamino, in whichthe alkyl groups are optionally substituted as described herein foralkyl. In certain embodiments, an amino group is —NHR^(N1), in whichR^(N1) is optionally substituted alkyl. Non-limiting examples of—NHR^(N1), in which R^(N1) is optionally substituted alkyl, include:optionally substituted alkylamino, a proteinogenic amino acid, anon-proteinogenic amino acid, a C₁₋₆ alkyl ester of a proteinogenicamino acid, and a C₁₋₆ alkyl ester of a non-proteinogenic amino acid.

The term “aminoalkyl,” as used herein, represents a chemical substituentof formula —R′—R″, where R′ is alkylene, and R″ is amino. Aminoalkyl maybe optionally substituted as defined for each of the two portions.

The term “aminoalkylaminocarbonyl,” as used herein, represents achemical substituent of formula R¹—R²—NH—(CO)—, where R¹ is amino, andR² is alkylene.

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one, two, or three (e.g., oneor two) aromatic rings and is exemplified by phenyl, naphthyl,1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl,indenyl, and the like, and may be optionally substituted with one, two,three, four, or five substituents independently selected from the groupconsisting of: (1) acyl; (2) alkyl; (3) alkenyl; (4) alkynyl; (5)alkoxy; (6) alkylsulfinyl; (7) aryl; (8) amino; (9) arylalkyl; (10)azido; (11) cycloalkyl; (12) cycloalkylalkyl; (13) cycloalkenyl; (14)cycloalkenylalkyl; (15) halo; (16) heterocyclyl (e.g., heteroaryl); (17)(heterocyclyl)oxy; (18) (heterocyclyl)aza; (19) hydroxy; (20) nitro;(21) thioalkoxy; (22) —(CH₂)_(q)CO₂R^(A), where q is an integer fromzero to four, and R^(A) is selected from the group consisting of (a)alkyl, (b) aryl, (c) hydrogen, and (d) arylalkyl; (23)—(CH₂)_(q)CONR^(B)R^(C), where q is an integer from zero to four andwhere R^(B) and R^(C) are independently selected from the groupconsisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl; (24)—(CH₂)_(q)SO₂R^(D), where q is an integer from zero to four and whereR^(D) is selected from the group consisting of (a) alkyl, (b) aryl, and(c) arylalkyl; (25) —(CH₂)_(q)SO₂NR^(E)R^(F), where q is an integer fromzero to four and where each of R^(E) and R^(F) is, independently,selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl,and (d) arylalkyl; (26) thiol; (27) aryloxy; (28) cycloalkoxy; (29)arylalkoxy; (30) heterocyclylalkyl (e.g., heteroarylalkyl); (31) silyl;(32) cyano; and (33) —S(O)R^(H) where R^(H) is selected from the groupconsisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl. Anunsubstituted aryl includes 6 to 14 carbon atoms (e.g., 6 to 10 carbonatoms). In some embodiments, each of these groups can be substitutedwith unsubstituted substituents as described herein for each respectivegroup.

The term “aryl alkyl,” as used herein, represents an alkyl groupsubstituted with an aryl group. Each of the aryl and alkyl portions maybe independently unsubstituted or substituted (e.g., optionallysubstituted aryl alkyl) as described for the individual groups.

The term “arylene,” as used herein, refers to a divalent group that isaryl, as defined herein, in which one hydrogen atom is replaced with avalency. Arylene may be unsubstituted or substituted (e.g., optionallysubstituted arylene) as described for aryl.

The term “arylcarbonyl,” as used herein, refers to a group —(CO)—R,where R is aryl. Arylcarbonyl may be unsubstituted or substituted (e.g.,optionally substituted arylcarbonyl) as described herein for aryl.

The term “aryloxy,” as used herein, refers to a group —OR, where R isaryl. Aryloxy may be unsubstituted or substituted (e.g., optionallysubstituted aryloxy) as described herein for aryl.

The term “aryloxy-carbonyl,” as used herein, refers to a group —COOR,where R is aryl. Aryloxycarbonyl may be unsubstituted or substituted(e.g., optionally substituted aryloxycarbonyl) as described herein foraryl.

The term “arylsulfonate,” as used herein, represents a group —S(O)₂—R,where R is aryl. Arylsulfonate may be unsubstituted or substituted(e.g., optionally substituted arylsulfonate) as described herein foraryl.

The term “aza,” as used herein, represents a divalent —N(R^(N1))— groupor a trivalent —N═ group. The aza group may be unsubstituted, whereR^(N1) is H or absent, or substituted, where R^(N1) is as defined for“amino.” Aza may also be referred to as “N,” e.g., “optionallysubstituted N.” Two aza groups may be connected to form “diaza.”

The term “azido,” as used herein, represents an N₃ group.

The term “carbamoyl,” as used herein, refers to a group of formulaRCOO—, where R is amino.

The term “carbocyclic,” as used herein, represents an optionallysubstituted C₃₋₁₂ monocyclic, bicyclic, or tricyclic structure in whichthe rings, which may be aromatic or non-aromatic, are formed by carbonatoms. Carbocyclic structures include cycloalkyl, cycloalkenyl, and arylgroups.

The term “cycloalkenyl,” as used herein, refers to a non-aromaticcarbocyclic group having from three to ten carbons (e.g., a C₃-C₁₀cycloalkylene), unless otherwise specified. Non-limiting examples ofcycloalkenyl include cycloprop-1-enyl, cycloprop-2-enyl,cyclobut-1-enyl, cyclobut-1-enyl, cyclobut-2-enyl, cyclopent-1-enyl,cyclopent-2-enyl, cyclopent-3-enyl, norbornen-1-yl, norbornen-2-yl,norbornen-5-yl, and norbornen-7-yl. The cycloalkenyl group may beunsubstituted or substituted (e.g., optionally substituted cycloalkenyl)as described for cycloalkyl.

The term “cycloalkenyl alkyl,” as used herein, represents an alkyl groupsubstituted with a cycloalkenyl group. Each of the cycloalkenyl andalkyl portions may be independently unsubstituted or substituted (e.g.,optionally substituted cycloalkenyl alkyl) as described for theindividual groups.

The term “cycloalkenylene,” as used herein, refers to a divalentcarbocyclic non-aromatic group having from three to ten carbons (e.g.,C₃-C₁₀ cycloalkenylene), unless otherwise specified. Non-limitingexamples of the cycloalkenylene include cycloprop-1-en-1,2-diyl;cycloprop-2-en-1,1-diyl; cycloprop-2-en-1,2-diyl;cyclobut-1-en-1,2-diyl; cyclobut-1-en-1,3-diyl; cyclobut-1-en-1,4-diyl;cyclobut-2-en-1,1-diyl; cyclobut-2-en-1,4-diyl; cyclopent-1-en-1,2-diyl;cyclopent-1-en-1,3-diyl; cyclopent-1-en-1,4-diyl;cyclopent-1-en-1,5-diyl; cyclopent-2-en-1,1-diyl;cyclopent-2-en-1,4-diyl; cyclopent-2-en-1,5-diyl;cyclopent-3-en-1,1-diyl; cyclopent-1,3-dien-1,2-diyl;cyclopent-1,3-dien-1,3-diyl; cyclopent-1,3-dien-1,4-diyl;cyclopent-1,3-dien-1,5-diyl; cyclopent-1,3-dien-5,5-diyl;norbornadien-1,2-diyl; norbornadien-1,3-diyl; norbornadien-1,4-diyl;norbornadien-1,7-diyl; norbornadien-2,3-diyl; norbornadien-2,5-diyl;norbornadien-2,6-diyl; norbornadien-2,7-diyl; and norbornadien-7,7-diyl.The cycloalkenylene may be unsubstituted or substituted (e.g.,optionally substituted cycloalkenylene) as described for cycloalkyl.

The term “cycloalkyl,” as used herein, refers to a cyclic alkyl grouphaving from three to ten carbons (e.g., a C₃-C₁₀ cycloalkyl), unlessotherwise specified. Cycloalkyl groups may be monocyclic or bicyclic.Bicyclic cycloalkyl groups may be of bicyclo[p.q.0]alkyl type, in whicheach of p and q is, independently, 1, 2, 3, 4, 5, 6, or 7, provided thatthe sum of p and q is 2, 3, 4, 5, 6, 7, or 8. Alternatively, bicycliccycloalkyl groups may include bridged cycloalkyl structures, e.g.,bicyclo[p.q.r]alkyl, in which r is 1, 2, or 3, each of p and q is,independently, 1, 2, 3, 4, 5, or 6, provided that the sum of p, q, and ris 3, 4, 5, 6, 7, or 8. The cycloalkyl group may be a spirocyclic group,e.g., spiro[p.q]alkyl, in which each of p and q is, independently, 2, 3,4, 5, 6, or 7, provided that the sum of p and q is 4, 5, 6, 7, 8, or 9.Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1]heptyl,2-bicyclo[2.2.1]heptyl, 5-bicyclo[2.2.1]heptyl, 7-bicyclo[2.2.1]heptyl,and decalinyl. The cycloalkyl group may be unsubstituted or substitutedas defined herein (e.g., optionally substituted cycloalkyl). Thecycloalkyl groups of this disclosure can be optionally substituted with:(1) acyl; (2) alkyl; (3) alkenyl; (4) alkynyl; (5) alkoxy; (6)alkylsulfinyl; (7) aryl; (8) amino; (9) arylalkyl; (10) azido; (11)cycloalkyl; (12) cycloalkylalkyl; (13) cycloalkenyl; (14)cycloalkenylalkyl; (15) halo; (16) heterocyclyl (e.g., heteroaryl); (17)(heterocyclyl)oxy; (18) (heterocyclyl)aza; (19) hydroxy; (20) nitro;(21) thioalkoxy; (22) —(CH₂)_(q)CO₂R^(A), where q is an integer fromzero to four, and R^(A) is selected from the group consisting of (a)alkyl, (b) aryl, (c) hydrogen, and (d) arylalkyl; (23)—(CH₂)_(q)CONR^(B)R^(C), where q is an integer from zero to four andwhere R^(B) and R^(c) are independently selected from the groupconsisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl; (24)—(CH₂)_(q)SO₂R^(D), where q is an integer from zero to four and whereR^(D) is selected from the group consisting of (a) alkyl, (b) aryl, and(c) arylalkyl; (25) —(CH₂)_(q)SO₂NR^(E)R^(F), where q is an integer fromzero to four and where each of R^(E) and R^(F) is, independently,selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl,and (d) arylalkyl; (26) thiol; (27) aryloxy; (28) cycloalkoxy; (29)arylalkoxy; (30) heterocyclylalkyl (e.g., heteroarylalkyl); (31) silyl;(32) cyano; and (33) —S(O)R^(H) where R^(H) is selected from the groupconsisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl. Insome embodiments, each of these groups can be substituted withunsubstituted substituents as described herein for each respectivegroup.

The term “cycloalkylene,” as used herein, refers to a divalent groupthat is cycloalkyl, as defined herein, in which one hydrogen atom isreplaced with a valency. Cycloalkylene may be unsubstituted orsubstituted (e.g., optionally substituted cycloalkylene) as describedfor cycloalkyl.

The term “cycloalkyl alkyl,” as used herein, represents an alkyl groupsubstituted with a cycloalkyl group. Each of the cycloalkyl and alkylportions may be independently unsubstituted or substituted (e.g.,optionally substituted cycloalkyl alkyl) as described for the individualgroups.

The term “dialkylamino,” as used herein, represents a group —N(R^(N1))₂,in which each RN′ is independently alkyl. Dialkylamino may be optionallysubstituted; each alkyl in optionally substituted dialkylamino isindependently and optionally substituted as described for alkyl.

The term “dialkylaminocarbonyl,” as used herein, represents a chemicalsubstituent of formula R—(CO)—, where R is dialkylamino.

The term “dialkylaminoalkylaminocarbonyl,” as used herein, represents achemical substituent of formula R¹—R²—NH—(CO)—, where R¹ isdialkylamino, and R² is alkylene.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, and fluorine.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens, or, when the halogengroup is F, haloalkyl group can be perfluoroalkyl. In some embodiments,the haloalkyl group can be further optionally substituted with 1, 2, 3,or 4 substituent groups as described herein for alkyl groups.

The term “heteroalkyl,” as used herein refers to an alkyl, alkenyl, oralkynyl group interrupted once by one heteroatom; twice, each time,independently, by one heteroatom; three times, each time, independently,by one heteroatom; or four times, each time, independently, by oneheteroatom. Each heteroatom is, independently, O, N, or S. In someembodiments, the heteroatom is O or N. An unsubstituted C_(X-Y)heteroalkyl contains from X to Y carbon atoms as well as the heteroatomsas defined herein. The heteroalkyl group may be unsubstituted orsubstituted (e.g., optionally substituted heteroalkyl). When heteroalkylis substituted and the substituent is bonded to the heteroatom, thesubstituent is selected according to the nature and valency of theheteroatom. Thus, the substituent, if present, bonded to the heteroatom,valency permitting, is selected from the group consisting of ═O,—N(R^(N2))₂, —SO₂OR^(N3), —SO₂R^(N2), —SOR^(N3), —COOR^(N3), anN-protecting group, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,cycloalkenyl, cycloalkynyl, heterocyclyl, or cyano, where each R^(N2) isindependently H, alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, orheterocyclyl, and each R^(N3) is independently alkyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, or heterocyclyl. Each of thesesubstituents may itself be unsubstituted or substituted withunsubstituted substituent(s) defined herein for each respective group.When heteroalkyl is substituted and the substituent is bonded to carbon,the substituent is selected from those described for alkyl, providedthat the substituent on the carbon atom bonded to the heteroatom is notCL, Br, or I. It is understood that carbon atoms are found at thetermini of a heteroalkyl group.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which include an aromatic ring systemthat contains at least one heteroatom. Thus, heteroaryls contain 4n+2 pielectrons within the mono- or multicyclic ring system. Heteroaryl can beunsubstituted or substituted (e.g., optionally substituted heteroaryl)with 1, 2, 3, or 4 substituents groups as defined for heterocyclyl.

The term “heteroarylcarbonyl,” as used herein, refers to a group—(CO)—R, where R is heteroaryl. Heteroarylcarbonyl may be unsubstitutedor substituted (e.g., optionally substituted heteroarylcarbonyl) asdescribed herein for heterocyclyl.

The term “heteroaryloxy,” as used herein, refers to a group —OR, where Ris heteroaryl. Heteroaryloxy may be unsubstituted or substituted (e.g.,optionally substituted heteroaryloxy) as described herein forheterocyclyl.

The term “heteroaryloxy-carbonyl,” as used herein, refers to a group—COOR, where R is heteroaryl. Heteroaryloxycarbonyl may be unsubstitutedor substituted (e.g., optionally substituted heteroaryloxycarbonyl) asdescribed herein for heterocyclyl.

The term “heteroaryl alkyl,” as used herein, represents an alkyl groupsubstituted with a heteroaryl group. Thus, heteroaryl alkyl is aheterocyclyl alkyl group, in which the heterocyclyl includes at leastone aromatic ring system including a heteroatom. Each of the heteroaryland alkyl portions may be independently unsubstituted or substituted(e.g., optionally substituted heteroaryl alkyl) as described for theindividual groups.

The term “heterocyclyl,” as used herein, represents a 5-, 6-, or7-membered ring or a fused ring system of two, three, or four rings,each of which is independently a 5-, 6-, or 7-membered ring, unlessotherwise specified, provided that at least one of the rings containsone, two, three, or four heteroatoms independently selected from thegroup consisting of nitrogen, oxygen, and sulfur. The 5-membered ringhas zero to two double bonds, and the 6- and 7-membered rings have zeroto three double bonds. An unsubstituted heterocyclyl contains from oneto twelve carbon atoms, unless specified otherwise. In some embodiments,an unsubstituted heterocyclyl contains at least two carbon atoms. Incertain embodiments, an unsubstituted heterocyclyl contains up to nicecarbon atoms. The fused “heterocyclyl” be a bridged multicyclicstructure in which one or more carbons and/or heteroatoms bridges twonon-adjacent members of a monocyclic ring, e.g., as found in aquinuclidinyl group. In some embodiments, the fused “heterocyclyl”includes bicyclic, tricyclic, and tetracyclic groups, in which at leastone of the rings includes one or more heteroatoms as defined herein, andthe remaining rings are carbocyclic rings, e.g., an aryl ring, acyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring. Non-limiting examples of such fused heterocyclylsinclude indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl, tropanes, and 1,2,3,5,8,8a-hexahydroindolizine.Non-limiting examples of heterocyclyls include pyrrolyl, pyrrolinyl,pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl,imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl,pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl,oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl,thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl,quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,furyl, thienyl, thiazolidinyl, isothiazolyl, isoindazoyl, triazolyl,tetrazolyl, oxadiazolyl, purinyl, thiadiazolyl (e.g.,1,3,4-thiadiazole), tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, dihydrothienyl, dihydroindolyl, tetrahydroquinolyl,tetrahydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl,benzofuranyl, benzothienyl and the like. Still other exemplaryheterocyclyls are: 2,3,4,5-tetrahydro-2-oxo-oxazolyl;2,3-dihydro-2-oxo-1H-imidazolyl; 2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl(e.g., 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl(e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl;2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Heterocyclic groups also include groups ofthe formula

whereF′ is selected from the group consisting of —CH₂—, —CH₂O— and —O—, andG′ is selected from the group consisting of —C(O)— and—(C(R′)(R″))_(v)—, where each of R′ and R″ is, independently, selectedfrom the group consisting of hydrogen or alkyl of one to four carbonatoms, and v is one to three and includes groups, such as1,3-benzodioxolyl, 1,4-benzodioxanyl, and the like. Any of theheterocyclyl groups mentioned herein may be optionally substituted withone, two, three, four, or five substituents independently selected fromthe group consisting of: (1) acyl; (2) alkyl; (3) alkenyl; (4) alkynyl;(5) alkoxy; (6) alkylsulfinyl; (7) aryl; (8) amino; (9) arylalkyl; (10)azido; (11) cycloalkyl; (12) cycloalkylalkyl; (13) cycloalkenyl; (14)cycloalkenylalkyl; (15) halo; (16) heterocyclyl (e.g., heteroaryl); (17)(heterocyclyl)oxy; (18) (heterocyclyl)aza; (19) hydroxy; (20) oxo; (21)nitro; (22) sulfide; (23) thioalkoxy; (24) —(CH₂)_(q)CO₂R^(A), where qis an integer from zero to four, and R^(A) is selected from the groupconsisting of (a) alkyl, (b) aryl, (c) hydrogen, and (d) arylalkyl; (25)—(CH₂)_(q)CONR^(B)R^(C), where q is an integer from zero to four andwhere R^(B) and R^(C) are independently selected from the groupconsisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl; (26)—(CH₂)_(q)SO₂R^(D), where q is an integer from zero to four and whereR^(D) is selected from the group consisting of (a) alkyl, (b) aryl, and(c) arylalkyl; (27) —(CH₂)_(q)SO₂NR^(E)R^(F), where q is an integer fromzero to four and where each of R^(E) and R^(F) is, independently,selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl,and (d) arylalkyl; (28) thiol; (29) aryloxy; (30) cycloalkoxy; (31)arylalkoxy; (31) heterocyclylalkyl (e.g., heteroarylalkyl); (32) silyl;(33) cyano; and (34) —S(O)R^(H) where R^(H) is selected from the groupconsisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl. Insome embodiments, each of these groups can be independentlyunsubstituted or substituted with unsubstituted substituent(s) asdescribed herein for each of the recited groups. For example, thealkylene group of an aryl-C₁-alkylene or a heterocyclyl-C₁-alkylene canbe further substituted with an oxo group to afford the respectivearyloyl and (heterocyclyl)oyl substituent group.

The term “heterocyclyl alkyl,” as used herein, represents an alkyl groupsubstituted with a heterocyclyl group. Each of the heterocyclyl andalkyl portions may be independently unsubstituted or substituted (e.g.,optionally substituted heterocyclyl alkyl) as described for theindividual groups.

The term “heterocyclylene,” as used herein, refers to a divalent groupthat is heterocyclyl, as defined herein, in which one hydrogen atom isreplaced with a valency. Heterocyclylene may be unsubstituted orsubstituted (e.g., optionally substituted heterocyclylene) as describedfor heterocyclyl.

The terms “hydroxyl” and “hydroxy,” as used interchangeably herein,represent an —OH group.

The term “internucleoside,” as used herein, refers to a position withinan oligonucleotide that is disposed between two contiguous nucleosides,one nucleoside and an adjacent abasic spacer, or two contiguous abasicspacers.

The term “n-membered ring,” as used herein, represents a cycloalkylene,arylene, or heterocyclylene having n atoms in a ring bearing bothvalencies. The n-membered rings can be unsubstituted or substituted(e.g., optionally substituted n-membered ring) as described herein forcycloalkyl, when n-membered ring is cycloalkylene, for aryl, whenn-membered ring is arylene, or for heterocyclyl, when n-membered ring isheterocyclylene.

The term “nitro,” as used herein, represents an —NO₂ group.

The term “nucleobase,” as used herein, represents a nitrogen-containingheterocyclic ring found at the 1′ position of the sugar moiety of anucleotide or nucleoside. Nucleobases can be unmodified or modified. Asused herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C orm5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808; those disclosed in The Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990; those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613; and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages289 302, (Crooke et al., ed., CRC Press, 1993). Nucleobases can be5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi etal., eds., Antisense Research and Applications 1993, CRC Press, BocaRaton, pages 276-278). These may be combined, in particular embodiments,with 2′-O-methoxyethyl sugar modifications. United States patents thatteach the preparation of certain of these modified nucleobases as wellas other modified nucleobases include, but are not limited to, the abovenoted U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091;5,614,617; and 5,681,941. For the purposes of this disclosure, “modifiednucleobases,” as used herein, further represents nucleobases, natural ornon-natural, which include one or more protecting groups as describedherein.

The term “nucleoside,” as used herein, represents a sugar-nucleobasecombination. Nucleoside, as used herein, is a compound, a monovalentgroup, or a divalent group. The sugar is: ribose, modified ribose (e.g.,2′-deoxyribose), mannose, arabinose, glucopyranose, galactopyranose,4-thioribose, a morpholino sugar (as found in morpholinooligonucleotides), threose (as found in threose nucleic acids),propanediol (as found in glycol nucleic acids), or a locked nucleic acid(e.g., ribose that is modified to include a bridge (e.g., a—CH₂—O-bridge), e.g., connecting 4′ and 2′ carbon atoms of the ribose).The sugar can be an L-sugar or D-sugar. A modified ribose has asubstitution at position 2′ with H, OR, R, halo (e.g., F), SH, SR, NH₂,NHR, NR₂, or CN, where R is an optionally substituted C₁₋₆ alkyl (e.g.,(C₁₋₆ alkoxy)-C₁₋₆-alkyl) or optionally substituted (C₆₋₁₄aryl)-C₁₋₄-alkyl. In some embodiments, the term “nucleoside” refers to agroup having the following structure:

in which B¹ is a nucleobase; R⁵ is H, halogen (e.g., F), O-protectedhydroxyl, or optionally substituted C₁₋₆ alkoxy (e.g., methoxy ormethoxyethoxy); Y¹ is H or C₁₋₆ alkyl (e.g., methyl); R⁶ is H or ahydroxyl protecting group; and each of 3′ and 5′ indicate the positionof a bond to another group. In some embodiments, the nucleoside is alocked nucleic acid (LNA). Locked nucleosides are known in the art andare described, for example, in U.S. Pat. Nos. 6,794,499; 7,084,125; and7,053,207. In certain embodiments, the nucleoside is a locked nucleicacid having the following structure:

-   -   in which    -   X is —O—, —S—, —N(R^(N)*)—, —C(R⁸R⁶*)—, —O—C(R⁷R⁷*)—,        —C(R⁶R⁶*)—O—, —S—C(R⁷R⁷*)—, —C(R⁶R⁶*)—S—, —N(RN*)—C(R⁷R⁷*)—,        —C(R⁶R⁶*)—N(RN*)—, or —C(R⁶R⁶*)—C(R⁷R⁷*)—;    -   B is a nucleobase;    -   R³* is a valency or OR^(A), where R^(A) is H or a hydroxyl        protecting group;

one or two pairs of non-geminal substituents selected from the groupconsisting of R¹*, R⁴*, R⁵, R⁵*, R⁶, R⁶*, R⁷, R⁷*, RN*, R², R²*, and R³combine to form one or two biradicals, respectively, where eachbiradical independently consists of 1-8 groups independently selectedfrom the group consisting of —C(R^(a)R^(b))—, —C(R^(a))═C(R^(a))—,—C(R^(a))═N—, —O—, —Si(R^(a))₂—, —S—, —SO₂—, —N(R^(a))—, and >C═Z, whereZ is selected from ═O—, ═S—, ═N(R^(a)), and ═CH₂, and each R^(a) andeach R^(b) is independently hydrogen, optionally substituted C₁₋₁₂alkyl, optionally substituted C₂₋₁₂ alkenyl, optionally substitutedC₂₋₁₂ alkynyl, —OH, C₁₋₁₂-alkoxy, C₂₋₁₂ alkenyloxy, —COOH, C₁₋₁₂alkoxycarbonyl, optionally substituted aryl, optionally substitutedaryloyl, optionally substituted aryloxy-carbonyl, optionally substitutedaryloxy, optionally substituted heteroaryl, optionally substitutedheteroaryloyl, optionally substituted heteroaryloxy-carbonyl, optionallysubstituted heteroaryloxy, amino, (C₁₋₆-alkyl)amino,di(C₁₋₆-alkyl)amino, carbamoyl, (C₁₋₆-alkyl)-amino-carbonyl,di(C₁₋₆-alkyl)-amino-carbonyl, amino-1-6-alkyl-aminocarbonyl,(C₁₋₆-alkyl)amino-1-6-alkyl-aminocarbonyl,di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, —NHCONH₂,C₂₋₇-alkanoylamino, C₁₋₆ alkanoyloxy, alkylsulfonyl, C₁₋₆alkylsulphonyloxy, nitro, azido, —SH, C₁₋₆ thioalkyl, or halo; and

-   -   each of the remaining substituents R¹*, R², R²*, R³, R⁴*, R⁵,        R⁵*, R⁶, R⁶*, R⁷, and R⁷* is independently hydrogen, optionally        substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl,        optionally substituted C₂₋₁₂ alkynyl, hydroxy, C₁₋₁₂ alkoxy,        C₂₋₁₂ alkenyloxy, —COOH, C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkanoyl,        formyl, optionally substituted aryl, optionally substituted        aryloxy-carbonyl, optionally substituted aryloxy, optionally        substituted arylcarbonyl, optionally substituted heteroaryl,        optionally substituted heteroaryloxy-carbonyl, optionally        substituted heteroaryloxy, optionally substituted        heteroarylcarbonyl, amino, (1-6-alkyl)amino,        di(C₁₋₆-alkyl)amino, carbamoyl, (C₁₋₆-alkyl)-amino-carbonyl,        di(C₁₋₆-alkyl)-amino-carbonyl, amino-1-6-alkyl-aminocarbonyl,        (C₁₋₆-alkyl)amino-1-6-alkyl-aminocarbonyl,        di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆        alkanoylamino, C₁₋₆ alkanoyloxy, C₁₋₆ alkylsulfonyl, C₁₋₆        alkylsulfonyloxy, nitro, azido, —SH, C₁₋₆-thioalkyl, or halogen;        or two remaining geminal substituents may combine to form ═O,        ═S, ═NR^(a), ═CH₂, or a 1-5 carbon atom(s) alkylene chain which        is optionally interrupted one or two heteroatoms independently        selected from the group consisting of —O—, —S—, and —(NR^(N))—,        where R^(N) is hydrogen or 014-alkyl; or two remaining vicinal        substituents combine to form an additional bond resulting in a        double bond; and R^(N)*, when present and not involved in the        biradical, is hydrogen or 014-alkyl.

In particular embodiments, the locked nucleic acid has the followingstructure:

In further embodiments, X is —O— and B is a nucleobase. In someembodiments,

-   -   R³* is a valency or OR^(A), where R^(A) is H or a hydroxyl        protecting group;    -   R²* and R⁴* combine to form a biradical consisting of 2-5        groups/atoms selected from —(CR*R*)_(r)—Y—(CR*R*)_(s)—,        —(CR*R*)_(l)—Y—(CR*R*)_(s)—Y—, —Y—(CR*R*)_(r+s)—Y—,        —Y—(CR*R*)_(l)—Y—(C R*R*)_(s)—, —(CR*R*)_(r+s)—, each R* is        independently hydrogen, halogen, —OH, —SH, amino, optionally        substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆ alkyl,        where Y is —O—, —S—, absent, or —N(R^(N))—, and each of r and s        is an integer from 0 to 4, provided that the sum r+s is 1-4, and        provided that, when the biradical is        —(CR*R*)_(r)—Y—(CR*R*)_(s)—, then Y is —S— or —N(R^(N′))—; and        each of the substituents R¹*, R², R³, R⁵, and R⁵* is        independently hydrogen, optionally substituted C₁₋₁₂ alkyl,        optionally substituted C₂₋₁₂ alkenyl, optionally substituted        C₂₋₁₂ alkynyl, —OH, C₁₋₁₂ alkoxy, C₂₋₁₂ alkenyloxy, —COOH, C₁₋₁₂        alkoxycarbonyl, C₁₋₁₂ alkanoyl, optionally substituted aryl,        optionally substituted aryloxy-carbonyl, optionally substituted        aryloxy, optionally substituted aryloyl, optionally substituted        heteroaryl, optionally substituted heteroaryloxy-carbonyl,        optionally substituted heteroaryloxy, optionally substituted        heteroaryloyl, amino, (C₁₋₆-alkyl)amino, di(C₁₋₆-alkyl)amino,        carbamoyl, (C₁₋₆-alkyl)-amino-carbonyl,        di(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,        (C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,        di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, —NHCONH₂,        C₂₋₇-alkanoylamino, C₁₋₆ alkanoyloxy, alkylsulfonyl, C₁₋₆        alkylsulphonyloxy, nitro, azido, —SH, C₁₋₆ thioalkyl, or halo.

The term “nucleotide,” as used herein, represents a nucleoside bonded toa phosphate, phosphorothioate, phosphorodithioate, phosphonate, orphosphoramidate.

The term “oligonucleotide,” as used herein, represents a compoundcontaining nucleosides and optionally abasic spacers covalently linkedto each other through internucleoside bridging groups, e.g., phosphates,phosphorothioates, phoshorodithioates, phosphites, phosphonates, andphosphoramidates. An oligonucleotide includes a total of 2-100nucleosides and abasic spacers, provided that the oligonucleotideincludes at least one nucleoside. In some embodiments, anoligonucleotide includes 1-6 (e.g., 1, 2, or 3) abasic spacers.

The terms “oxa” and “oxy,” as used interchangeably herein, represents adivalent oxygen atom that is connected to two groups (e.g., thestructure of oxy may be shown as —O—).

The term “oxo,” as used herein, represents a divalent oxygen atom thatis connected to one group (e.g., the structure of oxo may be shown as═O).

The term “pseudohalogen,” as used herein, represents an optionallysubstituted alkylsulfonate or optionally substituted arylsulfonate.Non-limiting examples of pseudohalogens include methanesulfonate,trifluoromethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

The term “protecting group,” as used herein, represents a group intendedto protect a functional group (e.g., a hydroxyl, an amino, or acarbonyl) from participating in one or more undesirable reactions duringchemical synthesis (e.g., polynucleotide synthesis). The term“O-protecting group,” as used herein, represents a group intended toprotect an oxygen containing (e.g., phenol, hydroxyl or carbonyl) groupfrom participating in one or more undesirable reactions during chemicalsynthesis. The term “N-protecting group,” as used herein, represents agroup intended to protect a nitrogen containing (e.g., an amino orhydrazine) group from participating in one or more undesirable reactionsduring chemical synthesis. Commonly used 0- and N-protecting groups aredisclosed in Greene, “Protective Groups in Organic Synthesis,” 3^(rd)Edition (John Wiley & Sons, New York, 1999), which is incorporatedherein by reference. Exemplary 0- and N-protecting groups includealkanoyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl,pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl,benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl,tri-iso-propylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyryl,phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and4-nitrobenzoyl. N-protecting groups useful for protection of amines innucleobases include phenoxyacetyl and (4-isopropyl)phenoxyacetyl.

Exemplary O-protecting groups for protecting carbonyl containing groupsinclude, but are not limited to: acetals, acylals, 1,3-dithianes,1,3-dioxanes, 1,3-dioxolanes, and 1,3-dithiolanes.

Other O-protecting groups include, but are not limited to: substitutedalkyl, aryl, and arylalkyl ethers (e.g., trityl; methylthiomethyl;methoxymethyl; benzyloxymethyl; siloxymethyl;2,2,2-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl;ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl;t-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl,p-methoxybenzyl, and nitrobenzyl); silyl ethers (e.g., trimethylsilyl;triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl;t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl;triphenylsilyl; and diphenymethylsilyl); carbonates (e.g., methyl,methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl;2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl;methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl).

Other N-protecting groups include, but are not limited to, chiralauxiliaries such as protected or unprotected D, L or D, L-amino acidssuch as alanine, leucine, phenylalanine, and the like;sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl,and the like; carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyl oxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropoxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups such as trimethylsilyl, and the like.Useful N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), phenoxyacetyl, and (4-isopropyl)phenoxyacetyl.

The term “silyl,” as used herein, refers to a group of formula —SiR₃,where each R is independently alkyl, alkenyl, aryl, or arylalkyl. Silylcan be optionally substituted in the same manner as defined for each Rgroup.

The term “sugar analogue,” as used herein, represents a C₃₋₆monosaccharide or C₃₋₆ alditol (e.g., glycerol), which is modified toreplace one hydroxyl group with a bond to an oxygen atom in formula(IIIA), (IIIB), (IIIC), or (IIID) (e.g., in formula (IVA), (IVB), (IVC),(IVD), (IVA′), (IVB′), (IVC′), (IVD′), (IVA″), (IVB″), (IVC″), or(IVD″)). A sugar analogue does not contain a nucleobase capable ofengaging in hydrogen bonding with a nucleobase in a complementarystrand. A sugar analogue is cyclic or acyclic. Further optionalmodifications included in a sugar analogue are: a replacement of one,two, or three of the remaining hydroxyl groups or carbon-bonded hydrogenatoms with H; optionally substituted C₁₋₆ alkyl; —(CH₂)_(t1)—OR^(Z),where t1 is an integer from 1 to 6, and R^(Z) is optionally substitutedC₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substitutedC₂₋₆ alkynyl, optionally substituted C₆₋₁₄ aryl, optionally substitutedC₃₋₈ cycloalkyl, optionally substituted (C₁₋₉ heterocyclyl)-C₁₋₆-alkyl,optionally substituted (C₆₋₁₀ aryl)-C₁₋₆-alkyl, or optionallysubstituted (C₃₋₈ cycloalkyl)-C₁₋₆-alkyl; introduction of one or twounsaturation(s) (e.g., one or two double bonds); and replacement of one,two, or three hydrogens or hydroxyl groups with substituents as definedfor alkyl, alkenyl, cycloalkyl, cycloalkenyl, or heterocyclyl.Non-limiting examples of sugar analogues are optionally substituted C₂₋₆alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₅cycloalkyl, optionally substituted C₅ cycloalkenyl, optionallysubstituted heterocyclyl (e.g., optionally substituted pyrrolidinyl,optionally substituted tetrahydrofuranyl, or optionally substitutedtetrahydrothiophenyl), optionally substituted (C₁₋₉heterocyclyl)-C₁₋₆-alkyl, or optionally substituted (C₃₋₈cycloalkyl)-C₁₋₆-alkyl.

The term “stereochemically enriched,” as used herein, refers to a localstereochemical preference for one enantiomer of the recited group overthe opposite enantiomer of the same group. Thus, a, oligonucleotidecontaining a stereochemically enriched phosphorothioate is anoligonucleotide, in which a phosphorothioate of predeterminedstereochemistry is present in preference to a phosphorothioate ofstereochemistry that is opposite of the predetermined stereochemistry.This preference can be expressed numerically using a diastereomericratio (dr) for the phosphorothioate of the predeterminedstereochemistry. The diastereomeric ratio for the phosphorothioate ofthe predetermined stereochemistry is the molar ratio of thediastereomers having the identified phosphorothioate with thepredetermined stereochemistry relative to the diastereomers having theidentified phosphorothioate with the stereochemistry that is opposite ofthe predetermined stereochemistry. The diastereomeric ratio for thephosphorothioate of the predetermined stereochemistry may be 75:25 orgreater (e.g., 80:20 or greater, 90:10 or greater, 95:5 or, or 98:2 orgreater).

The term “sulfide,” as used herein, represents —S— or ═S.

The term “thioalkyl,” as used herein, refers to a divalent group —SR, inwhich R is alkyl. Thioalkyl may be unsubstituted or substituted (e.g.,optionally substituted thioalkyl) as described for alkyl.

The term “thiocarbonyl,” as used herein, represents a C(═S) group.Non-limiting example of functional groups containing a “thiocarbonyl”includes thioesters, thioketones, thioaldehydes, thioanhydrides,thioacyl chlorides, thioamides, thiocarboxylic acids, andthiocarboxylates.

The term “thioheterocyclylene,” as used herein, represents a divalentgroup —S—R′—, where R′ is a heterocyclylene as defined herein.

The term “thiol,” as used herein, represents an —SH group.

One of skill in the art will recognize that references P-stereogenicgroups, compounds containing them, and diastereoselective synthesesutilizing the same are for enantioenriched and diastereoenrichedcompositions of the compounds (e.g., enantiomeric ratio of 90:10 orgreater (e.g., 95:5 or greater or 98:2 or greater)), where the majorstereoisomer is that which is identified either by a structure or by astereochemical identifier, such as (S) or (R) for the carbonstereocenters and (S_(P)) or (R_(P)) for the phosphorus stereocenters.

DETAILED DESCRIPTION

The invention provides P-stereogenic groups for diastereoselectivesynthesis of stereochemically enriched P-stereogenic compounds.P-stereogenic groups of the invention can be used in highlydiastereoselective synthesis of P-stereogenic phosphorothioates (e.g.,with dr of 90:10 or greater (e.g., 95:5 or greater or 98:2 or greater)).Advantageously, P-stereogenic groups (e.g., those having R³ and R⁴ beH)) can be readily accessed through a short (e.g., a two-step synthesis)from commercially available materials. A P-stereogenic group of theinvention is a group of formula (IA), (IB), (IC), or (ID):

-   -   where    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring; and    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.

In some embodiments, the P-stereogenic group is a group of formula (IA)or (IB).

In certain embodiments, the P-stereogenic group is of the followingstructure:

In particular embodiments, R³ and R⁴ are each H. In some embodiments,the P-stereogenic group is a group of formula (IIA), (IIB), (IIA′),(IIB′), (IIA″), or (IIB″).

The P-stereogenic groups of the invention may be provided in a compoundof formula (IIIA), (IIIB), (IIIC), or (IIID):

-   -   wherein    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   A is an optionally substituted C₁₋₁₂ alkyl, optionally        substituted C₃₋₁₀ cycloalkyl, optionally substituted C₃₋₁₀        cycloalkyl-C₁₋₆-alkyl, optionally substituted C₁₋₉ heterocyclyl,        optionally substituted C₁₋₉ heterocyclyl-C₁₋₆-alkyl, sugar        analogue, nucleoside, nucleotide, or oligonucleotide;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring; and    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.

In certain embodiments, the P-stereogenic group is of formula (IIIC′) or(IIID′):

In certain embodiments, the compound is of the following structure:

In particular embodiments, A is an optionally substituted C₁₋₁₂ alkyl,optionally substituted C₃₋₁₀ cycloalkyl, optionally substituted C₃₋₁₀cycloalkyl-C₁₋₆-alkyl, optionally substituted C₁₋₉ heterocyclyl,optionally substituted C₁₋₉ heterocyclyl-C₁₋₆-alkyl, or sugar analogue.

In further embodiments, A is a group of formula (X):

-   -   where    -   each of R^(A1), R^(A2), and R^(A3) is independently H, —OR^(A4),        or —N(R^(A4))(R^(A5)); where R^(A4) is optionally substituted        C₁₋₁₆ alkyl, optionally substituted C₂₋₁₆ heteroalkyl, or a        protecting group, and R^(A5) is H optionally substituted C₁₋₁₆        alkyl, optionally substituted C₂₋₁₆ heteroalkyl, or a protecting        group; and    -   each of m1, m2, m3, and m4 is independently an integer from 0 to        11, provided that the quaternary carbon in the structure above        is bonded to 0 or 1 atoms other than carbon and hydrogen, and        provided that the sum of m1, m2, m3 and m4 is 11 or less.

In some embodiments, the compound is of formula (IVA), (IVB), (IVA′),(IVB′), (IVA″), or (IVB″). In other embodiments, the phosphoramidite isof formula (IVE), (IVF), (IVE′), (IVF′), (IVE″), or (IVF″):

In certain embodiments, P-stereogenic groups may be provided innucleoside phosphoramidites. The nucleoside phosphoramidites of theinvention can be used to prepare oligonucleotides having P-stereogenicphosphorothioates with high diastereoselectivity (e.g., with dr of 90:10or greater (e.g., 95:5 or greater or 98:2 or greater)). Advantageously,nucleoside phosphoramidites of the invention (e.g., those having R³ andR⁴ be H) can be readily accessed through a short synthesis (e.g., atwo-step synthesis) from commercially available materials. Accordingly,the nucleoside phosphoramidites of the invention are a practicalsolution for high-yield synthesis of oligonucleotides havingstereochemically enriched P-stereogenic phosphorothioates.

The nucleoside phosphoramidites of the invention include a sugar bondedto a nucleobase and to a phosphoramidite of the following structure:

-   -   where    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring; and    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.

In certain embodiments, the phosphoramidite is of formula (VA) or (VB).In particular embodiments, the phosphoramidite is of formula (VC′) or(VD′):

In some embodiments, the nucleoside phosphoramidite is of the followingstructure:

-   -   where    -   B¹ is a nucleobase;    -   Y¹ is H or C₁₋₆ alkyl (e.g., methyl);    -   R⁵ is H, O-protected hydroxyl, optionally substituted C₁₋₆        alkoxy, or halogen (e.g., F); and    -   R⁶ is a hydroxyl protecting group;    -   and the remaining variables are as defined for formulas (VA),        (VB), (VC), and (VD).

In particular embodiments, the nucleoside phosphoramidite is of formula(VIC′) or (VID′):

In certain embodiments, the phosphoramidite is of the followingstructure:

In particular embodiments, R³ and R⁴ are each H. In some embodiments,the phosphoramidite is of formula (VIIA), (VIIB), (VIIA′), or (VIIB′).In other embodiments, the phosphoramidite is of formula (VIIE), (VIIF),(VIIE′), (VIIF′), (VIIE″), or (VIIF″):

Diastereoselective Preparation of Oligonucleotides ContainingPhosphorothioate Phosphodiester

The nucleoside phosphoramidites of the invention may be used for thediastereoselective preparation of oligonucleotides containing aphosphorothioate phosphodiester using reaction conditions known in theart for the phosphoramidite route for oligonucleotide synthesis.

Typically, a nucleoside phosphoramidite of formula (VA) produces aninternucleoside (R_(P))-phosphorothioate, and a nucleosidephosphoramidite of formula (VB) produces an internucleoside(S_(P))-phosphorothioate.

In a typical oligonucleotide chain growth step, a nucleosidephosphoramidite of the invention is coupled to a nucleoside having a5′-hydroxyl (e.g., a nucleoside linked to a solid support) or anoligonucleotide having a 5′-hydroxyl (e.g., an oligonucleotide linked toa solid support) to produce a product oligonucleotide including aninternucleoside phosphite substituted with a ring-opened chiralauxiliary. Typically, the coupling step is performed in the presence ofa coupling activator. Coupling activators are known in the art;non-limiting examples of coupling activators are(benzylthio)-1H-tetrazole (BTT), N-phenylimidazoliumtrifluoromethanesulfonate (PhIMT), 1-(cyanomethyl)pyrrolidiniumtrifluoromethanesulfonate (CMPT), 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU), 4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole,benzimidazolium trifluoromethanesulfonate (BIT), benzotriazole,3-nitro-1,2,4-triazole (NT), tetrazole, (ethylthio)-1H-tetrazole (ETT),(4-nitrophenyl)-1H-tetrazole, 1-(cyanomethyl)piperidiniumtrifluoromethanesulfonate, and N-cyanomethyldimethylammoniumtrifluoromethanesulfonate. In certain embodiments (e.g., when thenucleoside phosphoramidite includes 2′-deoxyribose), the couplingactivator is preferably CMPT. The product oligonucleotide may be anoligonucleotide (e.g., an oligonucleotide having a total of 2-100nucleosides (e.g., 2 to 50 or 2 to 35) including one or more (e.g., 1 to6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) internucleoside groupsindependently selected from the group consisting of linkers of formula(XIA) and (XIB):

where

-   -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring;    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl; and    -   R⁷ is acyl (e.g., alkanoyl).

The oligonucleotides including one or more internucleoside groups offormula (XIA) and/or (XIB) may be intermediates in the synthesis of anoligonucleotide including at least one stereochemically enrichedinternucleoside phosphorothioate. For example, these oligonucleotidesmay be subjected to a sulfurization reaction with a sulfurizing agent(e.g., Beaucage reagent;3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione(DDTT); S₈; or a compound of formula (XA) or (XB)) to produce anoligonucleotide (e.g., an oligonucleotide having a total of 2-100nucleosides (e.g., 2 to 50 or 2 to 35) including one or more (e.g., 1 to6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) internucleoside groupsindependently selected from the group consisting of linkers of formula(XIIA) and (XIIB):

where the variables are as describe for formulae (XIA) and (XIB).

Sulfurizing agents are known in the art; non-limiting examples of thesulfurizing agents are Beaucage reagent;3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione(DDTT); S₈; and compounds of formula (XA) and (XB).

The compound of formula (XA) is of the following structure:

R⁸—S—S—R⁸,   (XA)

-   -   or a salt thereof,    -   where    -   each R⁸ is independently R⁹C(X¹)—, (R¹⁰)₂P(X¹) or R¹¹S(O)₂—,        where each R⁹ is independently alkylamino or dialkylamino; each        R¹⁰ is independently alkoxy or aryloxy; each R¹¹ is        independently hydroxyl, alkyl, aryl, or heteroaryl; and X¹ is ═O        or ═S.

The compound of formula (XB) is of the following structure:

-   -   where    -   X² is O or S; and    -   R¹² is aryl, amino, or alkoxy.

For example, the compound of formula (XB) can be:

The oligonucleotide including one or more internucleoside groups offormula (XIIA) and/or (XIIB) is then fed back into the synthesis, e.g.,by deprotecting the 5′-protecting group and treating the resulting5′-hydroxyl as described above or using a different nucleosidephosphoramidite (e.g., those known in the art). Alternatively, if thesynthesis of the oligonucleotide chain is complete, the oligonucleotidemay be subjected to further modifications (e.g., capping the 5′ end). Ifthe oligonucleotide chain is linked through a linker to solid support,the linker may be cleaved using methods known in the art after thesynthesis of the oligonucleotide chain is complete. The remainder of thering-opened chiral auxiliaries of the invention may be removed fromphosphotriesters through hydrolysis with aqueous ammonia (30% (w/w))(e.g., by heating for 12-24 hours at, e.g., about 55° C.). The remainderof the ring-opened chiral auxiliaries of the invention may be removedbefore, after, or concomitantly with the oligonucleotide chain removalfrom the solid support.

A non-limiting example of an oligonucleotide synthesis route is shown inScheme 1.

As shown in Scheme 1, compound A, which is a protected nucleosideoptionally linked to a solid support, may be subjected to a deprotectionreaction to remove the 0-protecting group (e.g., DMT) at R⁶ and producecompound B. Compound B is then coupled to phosphoramidite C to producephosphite D. In certain embodiments (e.g., when the nucleosidephosphoramidite includes 2′-deoxyribose, e.g., when R⁵ is H), thecoupling activator is preferably CMPT.

Compound D is oxidized using a sulfurizing agent to affordphosphorothioate E with retention of stereochemistry.

Nucleoside phosphoramidites including phosphoramidites of formula (IA),(IB), (IC), and (ID) can be in the synthesis of oligonucleotides inaccordance with the procedure described above using reaction conditionsknown in the art.

Preparation of Nucleoside Phosphoramidites Phosphoramidite Precursors

The nucleoside phosphoramidites of the invention may be prepared from acompound of formula:

-   -   where    -   is a single carbon-carbon bond or a double carbon-carbon bond;    -   X is a halogen (e.g., Cl or Br) or pseudohalogen;    -   each of R¹ and R² is independently an optionally substituted        C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R²,        together with the atoms to which each is attached, combine to        form an optionally substituted 5- to 8-membered ring; and    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.

In particular embodiments, the precursor to a nucleoside phosphoramiditemay be a compound of formula:

-   -   where    -   each of R³ and R⁴ is independently H, optionally substituted        C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl; and    -   X is halogen (e.g., Cl or Br) or pseudohalogen.

A non-limiting example of the preparation of a nucleosidephosphoramidite of the invention is shown in Scheme 2.

As shown in Scheme 2, aminoalcohol G can be converted tooxazaphospholane of formula (VIIIA) using an electrophilic source ofphosphorus (III), e.g., phosphorus (III) halide (e.g., PCl₃). Anoxazaphospholane of formula (VIIIA) may be coupled to nucleoside H togive a nucleoside phosphoramidite of formula (VIA). The reactionconditions useful for this coupling are known in the art and typicallyinvolve the use of a sterically hindered organic base (e.g.,N,N-diisopropylethylamine). Typically, the oxazaphospholane formationand phosphoramidite formation are performed in a one-pot transformationwithout isolation or purification of the oxazaphospholane of formula(VIIIA).

Nucleoside phosphoramidites including phosphoramidites of formula (VA),(VB), (VC), and (VD) can be prepared according to the proceduredescribed above using reaction conditions known in the art.

Aminoalcohol G and its enantiomer can be prepared from the correspondingamino acid using methods and reactions known in the art. Aminoalcohol Gcan be used in the preparation of compounds containing a P-stereogenicgroup of formula (IA) or (IB) (e.g., compounds of formula (IIIA) or(IIIB)). Aminoalcohol I and its enantiomer for the preparation ofphosphoramidites of formula (VC) and (VD) can be prepared from thecorresponding amino acids using methods and reactions known in the art.Aminoalcohol I can be used in the preparation of compounds containing aP-stereogenic group of formula (IC) or (ID) (e.g., compounds of formula(IIIC) or (IIID)). Aminoalcohol I is a compound of the followingstructure:

where each of R¹ and R² is independently optionally substituted C₁₋₆alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ and R², together withthe atoms to which each is attached, combine to form an optionallysubstituted 5- to 8-membered ring.

Advantageously, when R³ and R⁴ are each H, nucleoside phosphoramiditesof the invention can be prepared through a short reaction sequence ofonly three steps, two of which can be carried out in one pot.

The following examples are meant to illustrate the invention. They arenot meant to limit the invention in any way.

EXAMPLES Chiral Auxiliaries and Phosphoramidites

To a solution of (2S)-dihydro-1H-indole-2-carboxylic acid 1 (1.63 g,10.0 mmol) in ether (50 mL) was added a solution of LiAlH₄ in THF (2M,7.5 mL, 15.0 mmol) under argon, and the mixture was stirred overnight.After completion of the reaction, the mixture was quenched withNa₂SO₄.10H₂O. The solid was filtered off and washed with ethyl acetate,and the filtrate was dried over anhydrous Na₂SO₄. The mixture wasfiltered, and the solvent evaporated to give a residue, which wassubjected to flash silica gel column purification on an ISCO(hexane/ethyl acetate, 10-70%) to give 1.44 g (96%) of compound 2 as agray solid. ¹H NMR (500 MHz, CDCl₃; ppm): δ7.10 (1H, d, J 7.5 Hz), 7.04(1H, t, J 7.5 Hz), 6.75 (1H, t, J 7.5 Hz), 6.69 (1H, d, J 7.5 Hz),4.10-4.06 (2H, m), 3.75 (1H, dd, J 11.0, 4.0 Hz), 3.60 (1H, dd, J 11.0,6.0 Hz), 3.12 (1H, dd, J 16.0, 9.0 Hz), 2.87 (1H, dd, J 16.0, 8.0 Hz);ESI MS for C₉H₁₁NO calculated 149.2, observed [M+H]⁺ 150.1.

To a solution of compound 2 (1.0 g, 6.7 mmol) in anhydrous THF (5 mL)was added N,N-diisopropylethylamine (2.41 mL, 13.4 mmol) under argon.The resulting mixture was added dropwise to a solution of phosphorustrichloride (0.58 mL, 6.7 mmol) in anhydrous THF (8 mL) at 0° C. underargon. The mixture was warmed to room temp and stirred for 1.5 h. In aseparate round bottom flask, a solution of5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-uridine (2.25 g, 4.0 mmol) andN,N-diisopropylethylamine (4.81 mL, 26.8 mmol) in THF (5 mL) under argonwas cooled to −78° C., and the above mixture was slowly added. Themixture was warmed to room temp, stirred for 3 h, diluted withdichloromethane (30 mL), and washed with saturated aqueous sodiumbicarbonate (20 mL). The organic layer dried over anhydrous sodiumsulfate and filtered. The filtrate was evaporated to afford a residue,which was subjected to flash silica gel amine column purification on anISCO (1-8% methanol in dichloromethane) to give 1.14 g (39%) of thetitle compound 3 as a white foam. ESI MS for C₄₀H₄₀N₃O₉P Calculated737.7, Observed 738.2 (M+1); ³¹ P NMR (202 MHz, CDCl₃): δ141.2 (s).

Compound 4 was prepared by the same procedure as reported here forCompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-uridine as astarting material. Compound 4 was produced in 24% yield. ESI MS forC₃₉H₃₇FN₃O₈P Calculated 725.7, Observed 748.3 (M+Na); ³¹ P NMR (202 MHz,CDCl₃): δ141.0 (s).

To a solution of (2S,3aS,7aS)-octahydro-1H-indole-2-carboxylic acid 5(1.69 g, 10.0 mmol) in ether (50 mL) was added a solution of LiAlH₄ inTHF (2M, 7.5 mL, 15 mmol) under argon, and the mixture was stirredovernight. After completion of the reaction, the mixture was quenchedwith Na₂SO₄.10H₂O, and the solids were filtered off and washed withethyl acetate. The filtrate was dried over anhydrous Na₂SO₄ andevaporated to give 1.23 g (79%) of the crude compound 6 as a colorlessoil. ¹H NMR (500 MHz, CDCl₃; ppm): δ3.70 (1H, dd, J 11.0, 3.5 Hz), 3.60(1H, dd, J 11.0, 6.0 Hz), 3.50-3.40 (1H, m), 3.24 (1H, q, J 6.0 Hz),2.13-2.08 (1H, m), 1.94-1.86 (1H, m), 1.75-1.65 (1H, m), 1.65-1.40 (6H,m), 1.35-1.23 (2H, m); ESI MS for C₉H₁₁NO calculated 155.2, observed[M+H]⁺ 156.1.

Compound 7 was prepared by the same procedure as reported here forCompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-uridine as astarting material. ESI MS for C₃₉H₄₃FN₃O₈P calculated 731.7, observed732.2 (M+1); ³¹ P NMR (202 MHz, CDCl₃): δ140.7 (s).

Compound 8 was prepared by the same procedure as reported here forCompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-uridine as astarting material. ESI MS for C₄₀H₄₆N₃O₉P calculated 743.8, observed742.5 (M−1); ³¹ P NMR (202 MHz, CDCl₃): δ140.0 (s).

To a solution of (2R)-dihydro-1H-indole-2-carboxylic acid 9 (4.90 g,30.0 mmol) in ether (100 mL) was added a solution of LiAlH₄ in THF (2M,22.5 mL, 45 mmol) under argon, and the mixture was stirred overnight.After completion of the reaction, the reaction mixture was quenched withNa₂SO₄.10H₂O, and the solids were filtered off and washed with ethylacetate. The filtrate was dried over anhydrous Na₂SO₄, the mixture wasfiltered, and the solvent evaporated to give a residue, which wassubjected to flash silica gel column purification on an ISCO(hexane/ethyl acetate, 10-70%) to give 3.68 g (82%) of compound 10 as agray solid. ¹H NMR (500 MHz, CDCl₃; ppm): δ7.10 (1H, d, J 7.5 Hz), 7.04(1H, t, J 7.5 Hz), 6.75 (1H, t, J 7.5 Hz), 6.69 (1H, d, J 7.5 Hz),4.10-4.06 (1H, m), 3.75 (1H, dd, J 11.0, 4.0 Hz), 3.60 (1H, dd, J 11.0,6.0 Hz), 3.12 (1H, dd, J 16.0, 9.0 Hz), 2.87 (1H, dd, J 16.0, 8.0 Hz);ESI MS for C₉H₁₁NO calculated 149.2, observed [M+H]⁺ 150.1.

Compound 11 was prepared by the same procedure as reported here forCompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-uridine as astarting material. Compound 11 was produced in 56% yield. ESI MS forC₄₀H₄₀N₃O₉P calculated 737.7, observed 738.2 (M+1); ³¹ P NMR (202 MHz,CDCl₃): δ141.3 (s).

Compound 12 was prepared by the same procedure as reported here forCompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-uridine as astarting material. Compound 12 was produced in 72% yield. ESI MS forC₃₉H₃₇FN₃O₈P Calculated 725.7, Observed 748.3 (M+Na); ³¹P NMR (202 MHz,CDCl₃): δ141.8 (s).

Compound 13 was prepared by the same procedure as reported here forCompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-cytidine(N-acetyl) as a starting material. Compound 13 was produced in 33%yield. ESI MS for C₄₁H₄₁N₄O₈P Calculated 748.8, Observed 747.4 (M-1);³¹P NMR (202 MHz, CDCl₃): δ140.2 (s).

Compound 14 was prepared by the same procedure as reported here forCompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-cytosine(N-acetyl) as a starting material. Compound 14 was produced in 27%yield. ESI MS for C₄₁H₄₁N₄O₈P Calculated 748.8, Observed 747.4 (M-1);³¹P NMR (202 MHz, CDCl₃): δ139.7 (s).

Compound 15 was prepared by the same procedure as reported here forCompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-cytosine(N-acetyl) as a starting material. Compound 15 was produced in 35%yield. ESI MS for C₄₂H₄₃N₄O₈P Calculated 778.8, Observed 779.3 (M); ³¹ PNMR (202 MHz, CDCl₃): δ141.0(s).

Compound 16 was prepared by the same procedure as reported here forCompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-adenosine(N-benzoyl) as a starting material. Compound 16 was produced in 48%yield. ESI MS for C₄₈H₄₅N₆O₈P Calculated 864.8, Observed 865.3 (M); ³¹ PNMR (202 MHz, CDCl₃): δ140.0 (s).

Compound 17 was prepared by the same procedure as reported here forcompound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-guanosine(N-i-butyryl) as a starting material. Compound 17 was produced in 10%yield. ESI MS for C₄₅H₄₇N₆O₉P Calculated 846.9, Observed 847.3 (M); ³¹ PNMR (202 MHz, CDCl₃): δ138.9 (s).

Compounds listed in Table 1 were prepared by the same procedure asreported here for compound 3.

TABLE 1 Che- mical ³¹P NMR Yield (202 Compound (%) MHz)

33 —

26 —

34 δ138.84 (s)

33 δ138.53 (s)

57 δ138.90 (s)

44 δ139.58 (s)

45 δ138.63 (s)

42 δ139.41 (s)

32 δ140.93 (s)

14 δ139.13 (s) iBu in this table stands for isobutyryl.

Synthesis of the Polynucleotide Constructs

All the polynucleotide constructs synthesized were modified at the2′-ribose sugar position with 2′-F, 2′-OMe, or 2′-deoxy modification.0-protecting groups, such as 2′-OTBDMS, can also be used. Automatedpolynucleotide synthesis (1 μmol scale) was carried out with thefollowing reagents/solvents:

-   -   Solid support—CPG Glen Uny support    -   Coupling agent—0.25 M BTT in acetonitrile    -   Oxidizer—0.02 M 12 in THF/Pyridine/H₂O (2×30 s oxidation per        cycle)    -   Deblock—3% Trichloroacetic Acid/DCM (2×40 s deblocks per cycle)    -   Cap Mix A—THF/2, 6-Lutidine/Ac₂O (2×30 s capping per cycle)    -   Cap Mix B—16% Methyl imidazole in THF (2×30 s capping per cycle)    -   Sulfurization—0.05 M sulfurizing reagent,        3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione        (DDTT), in 60% pyridine/40% acetonitrile (3×60 s sulfurization        per cycle)    -   Coupling—Phosphoramidites were suspended to a concentration of        100 mM in anhydrous acetonitrile prior to synthesis,        phosphoramidite activation was performed with 2.5-fold molar        excess of BTT, 0.25 M in acetonitrile. Activated        phosphoramidites were coupled for 3×60 seconds per cycle

Polynucleotide Deprotection and Purification Protocol:

-   -   When polynucleotides contain standard nucleobase protecting        groups (such as A-Bz, C-Ac and G-iBu etc.), the following        cleavage and deprotection conditions were used:    -   polynucleotides were cleaved and deprotected in 1.0 mL of AMA        (1:1 ratio of 36% aq. ammonia and 40% methylamine in methanol)        for 2 h at room temperature followed by centrifugal evaporation.    -   Crude polynucleotide pellets were re-suspended in 100 μL of 50%        acetonitrile/water, briefly heated to 65° C., and vortexed        thoroughly. Total 100 μL crude polynucleotide samples were        injected onto reverse phase HPLC with the following        buffers/gradient:        -   Buffer A=50 mM aqueous triethylammonium acetate (TEAA)        -   Buffer B=90% acetonitrile in water        -   Flow Rate=1 mL/min        -   Gradient:            -   0-2 min (100% Buffer A/0% Buffer B)            -   2-42 min (0% to 60% Buffer B)            -   42-55 min (60% to 100% Buffer B)    -   Across the dominant reverse phase HPLC peaks, 0.5 mL fractions        were collected and analyzed by MALDI-TOF mass spectrometry to        confirm the presence of compounds with the desired mass peaks.        Purified fractions containing compounds with the correct mass        peaks were frozen and lyophilized. Once dry, fractions were        re-suspended, combined with corresponding fractions, frozen, and        lyophilized to give the final product.

Polynucleotides requiring additional deprotection were initiallyisolated as described above followed by the necessary secondarydeprotection steps (see below):

Secondary Deprotection of Polynucleotides Having TBDMS Protection:

Reverse phase HPLC-purified polynucleotide products were re-suspended in219 μL of anhydrous DMSO, heated briefly to 65° C., and vortexedthoroughly. To the DMSO solution, 31 μL of 6.1 M triethylaminetrihydrofluoride (TEA.3HF) was added to give a final concentration of0.75 M. The reaction was allowed to proceed at room temperature for ˜1 hper TBDMS-protected hydroxyl modification. Reaction was monitored byMALDI-TOF mass spectrometry to confirm complete deprotection. Oncedeprotection was complete, 35 μL of 3M sodium acetate and 1 mL ofbutanol were sequentially added. Samples were vortexed thoroughly andplaced at −80° C. for 2 h. After 2 h, samples were centrifuged to pelletthe polynucleotides. The butanol layer was removed, and thepolynucleotide pellet was re-suspended in 1 mL of aqueous 20%acetonitrile. Samples were gel-filtered for isolation of the finalpolynucleotide construct.

Synthesis of Polynucleotide Constructs with Stereochemically EnrichedInternucleoside Phosphorothioates (PS):

The following modified experimental conditions have been used for thesynthesis of polynucleotide constructs including stereochemicallyenriched internucleoside phosphorothioates from chiral phosphoramiditemonomers. Automated polynucleotide synthesis (1 μmol scale) was carriedout with the following reagents/solvents:

-   -   Solid support—CPG Glen Uny support    -   Coupling agent—BTT/ETT/CMPT/phenyl imidazole as required    -   Oxidizer—0.02 M 12 in THF/Pyridine/H₂O (2×30 s oxidation per        cycle)    -   Deblock—3% dichloroacetic Acid/DCM (2×40 s deblocks per cycle)    -   Cap Mix A—THF/2,6-lutidine/Ac₂O (2×30 s capping per cycle)    -   Cap Mix B—16% methyl imidazole in THF (2×30 s capping per cycle)    -   Sulfurization—0.05 M sulfurizing reagent,        3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione        (DDTT), in 60% pyridine/40% acetonitrile (3×120 s sulfurization        per cycle)    -   Coupling—chiral phosphoramidites (e.g., compounds of formula        (IIA) or (IIB)) were suspended to a concentration of 100 mM in        anhydrous acetonitrile prior to synthesis, phosphoramidite        activation was performed with 2.5-fold molar excess of        respective activators as specified (BTT=0.25 M in acetonitrile,        CMPT=0.5 M in acetonitrile, Ph-Imidazole=0.5 M in Acetonitrile).        Activated chiral phosphoramidites were coupled for 3×200 seconds        per cycle.

Chiral Polynucleotide Deprotection and Purification Protocol:

-   -   Following automated polynucleotide synthesis, stereopure        phosphorothioate containing polynucleotides with standard        nucleobase protecting groups (such as A-Bz, C-Ac, and G-iBu        etc.) and chiral auxiliary were deprotected and cleaved with the        following conditions: DMT protected chiral phosphorothioate        polynucleotides on solid support was suspended in 1.0 mL of        aqueous ammonia (30 wt %) and heated at 55° C. for 12-24 h,        followed by centrifugal evaporation.    -   Crude chiral polynucleotide pellets were re-suspended in 100 μL        of 50% acetonitrile, briefly heated to 65° C., and vortexed        thoroughly. Total 100 μL crude polynucleotide samples were        injected onto reverse phase HPLC with the following        buffers/gradient:        -   Buffer A=100 mM aqueous triethylammonium acetate (TEAA)        -   Buffer B=90% acetonitrile in water        -   Flow Rate=1 mL/min        -   Gradient:            -   0-2 min (100% Buffer A/0% Buffer B)            -   2-50 min (0% to 45% Buffer B)            -   50-55 min (45% to 100% Buffer B)    -   Across the dominant reverse phase HPLC peaks, 1.0 mL fractions        were collected and analyzed by MALDI-TOF mass spectrometry to        confirm the presence of compounds with the desired mass peaks.        Purified fractions containing compounds with the correct mass        peaks were frozen and lyophilized. Once dry, fractions were        re-suspended, combined with corresponding fractions, frozen, and        lyophilized to give the final product.

Analysis of Stereochemical Purity of Phosphorothioate ContainingPolynucleotides:

DMT protected oligonucleotides with stereochemically enrichedphosphorothioates were analyzed by HPLC/UPLC to determine thediastereoselectivity of R_(P) and S_(P) isomers. The absolutestereochemical identity of the internucleoside phosphorothioateidentified with an asterisk (*) was determined through comparison of theHPLC traces of the oligonucleotides of the invention to the HPLC tracesof authentic racemic and diastereomerically enriched oligonucleotidesthat were prepared using methods known in the art. The HPLC conditionswere as follows:

-   -   Reverse Phase HPLC    -   Column: AdvancedBio Oligonucleotide, 2.1×100 mm, 2.7 μm    -   Mobile Phase A: 100 mM tetraethylammonium acetate in water    -   Mobile Phase B: acetonitrile    -   Gradient: 10-12% mobile phase B in 45 min    -   Column Temperature: 60° C.    -   Flow Rate: 0.35 mL/min    -   Detection: 260 nm (UV)

For comparison, reference standards of the same oligonucleotide withR_(P) and S_(P) isomers were prepared using literature methods asdescribed elsewhere (Oka et al., Chem. Soc. Rev., 40:5829-5843, 2011;Oka et al., Org. Lett., 11:967-970, 2009; and U.S. pre-grant publicationNos. 2013/0184450 and 2015/0197540).

Diastereomer ratios (S_(P):R_(P)) have been established by integratingthe product peaks in UPLC traces of the prepared oligonucleotides.Absolute stereochemical identity of the dominant diastereomer wasdetermined by comparison to the reference standard. UPLC was performedas follows. Samples were dissolved in water, injected onto UPLC, andanalyzed under the following conditions:

-   -   Column: Xbridge C18, 4.6×150 mm, 5 μm    -   Mobile phase A=50 mM aqueous triethylammonium acetate (TEAA) in        water    -   Mobile phase B=Acetonitrile in water    -   Flow Rate=1 mL/min    -   Column Temperature=50° C.    -   Detection=260 nm    -   Gradient:        -   0-1 min (90% mobile phase A/10% mobile phase B)        -   1-30 min (88.5% mobile phase A/11.5% mobile phase B)

The stereochemical purity, stereochemical identity, and couplingactivators used in the synthesis of the prepared oligonucleotides areshown in Table 2.

TABLE 2 Oligonu- En- cleotide Acti- try (5′-3′) vator PN (**) Sp:Rp 1uUGAAGUAAA BTT Racemic Racemic Racemic 2 u*UGAAGUAAA BTT (R_(P))-OHI(S_(P)) >99.0:<1.0 3 u*UGAAGUAAA BTT (R_(P))-DHI (S_(P)) >99.0:<1.0 4u*UGAAGUAAA PhIMT (R_(P))-OHI (S_(P)) >99.0:<1.0 5 u*UGAAGUAAA PhIMT(R_(P))-DHI (S_(P)) >99.0:<1.0 6 u*UGAAGUAAA CMPT (R_(P))-OHI(S_(P)) >99.0:<1.0 7 u*UGAAGUAAA CMPT (R_(P))-DHI (S_(P)) >99.0:<1.0 8uUGAAGUAAA BTT Racemic Racemic Racemic 9 u*UGAAGUAAA BTT (R_(P))-OHI(S_(P)) 91:11 10 u*UGAAGUAAA BTT (R_(P))-DHI (S_(P)) >99.0:<1.0 11u*UGAAGUAAA CMPT (R_(P))-OHI (S_(P)) 95.0:5.0 12 u*UGAAGUAAA CMPT(R_(P))-DHI (S_(P)) >99.0:<1.0 13 u*UGAAGUAAA PhIMT (R_(P))-OHI(S_(P)) >99.0:<1.0 14 u*UGAAGUAAA PhIMT (R_(P))-DHI (S_(P)) >99.0:<1.015 u*UGAAGUAAA BTT (S_(P))-DHI (R_(P)) 18:82 16 u*UGAAGUAAA BTT(S_(P))-DHI (R_(P)) <1:>99.0 17 u*UGAAGUAAA CMPT (S_(P))-DHI (R_(P))1.7:98.3 18 u*UGAAGUAAA CMPT (S_(P))-DHI (R_(P)) <1:>99.0 19 u*UGAAGUAAAPhIMT (S_(P))-DHI (R_(P)) 4.2:95.8 20 u*UGAAGUAAA PhIMT (S_(P))-DHI(R_(P)) 7.4:92.6 21 mNUAAGUAAA BTT Racemic Racemic Racemic 22m*NUAAGUAAA BTT (S_(P))-DHI (R_(P)) 16:84 23 m*NUAAGUAAA BTT (R_(P))-DHI(S_(P)) 73.9:26.1 24 m*NUAAGUAAA CMPT (S_(P))-DHI (R_(P)) <1.0:>99.0 25m*NUAAGUAAA CMPT (R_(P))-DHI (S_(P)) 96.3:3.7 26 m*NUAAGUAAA PhIMT(S_(P))-DHI (R_(P)) 12.1:87.9 27 m*NUAAGUAAA PhIMT (R_(P))-DHI (S_(P))74.4:25.6 28 aUGAAGUAAA BTT Racemic Racemic Racemic 29 a*UGAAGUAAA CMPT(S_(P))-DHI (R_(P)) <1.0:>99.0 30 mUGAAGUAAA BTT Racemic Racemic Racemic31 m*UGAAGUAAA CMPT (S_(P))-DHI (R_(P)) <1.0:>99.0 30 gUGAAGUAAA BTTRacemic Racemic Racemic 31 g*UGAAGUAAA CMPT (S_(P))-DHI (R_(P))<1.0:>99.0 32 aNUAAGUAAA BTT Racemic Racemic Racemic 33 a*NUAAGUAAA CMPT(R_(P))-DHI (S_(P)) 98.4:1.6 34 gNUAAGUAAA BTT Racemic Racemic Racemic35 g*NUAAGUAAA CMPT (R_(P))-DHI (S_(P)) 90.9:9.1 36 tNUAAGUAAA BTTRacemic Racemic Racemic 37 t*NUAAGUAAA CMPT (R_(P))-DHI (S_(P))86.8:13.2

In Table 2, lower case u is uridine having 2′-F and a 3′ position bondedto phosphorothioate; lower case bold u is uridine having 2′-OMe and a 3′position bonded to phosphorothioate; lower case a is 2′-deoxyadenosinehaving a 3′ position bonded to phosphorothioate; lower case bold a isadenosine having a 2′-OMe and a 3′ position bonded to phosphorothioate;lower case g is 2′-deoxyguanosine having a 3′ position bonded tophosphorothioate; lower case bold g is guanosine having 2′-OMe and a 3′position bonded to phosphorothioate; lower case m is 2′-deoxycytidinehaving a 3′ position bonded to phosphorothioate; lower case bold m iscytidine having 2′-OMe and a 3′ position bonded to phosphorothioate;lower case t is 2′-deoxythymidine having a 3′ position bonded tophosphorothioate; * indicates a stereochemically enrichedinternucleoside phosphorothioate; UPPER CASE LETTERS identifynucleosides having 2′-F and a 3′ position bonded to phosphate; UPPERCASE BOLD LETTERS identify nucleosides having 2′-OMe and a 3′ positionbonded to phosphate; N is a 2′-deoxyguanosine having a 3′ positionbonded to phosphate; PN means phosphoramidite; (**) providesstereochemical identity of the internucleoside phosphorothioateidentified with * in the oligonucleotide column; and DHI and OHIrepresent the following structures:

respectively.

OTHER EMBODIMENTS

Various modifications and variations of the described invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the invention.

Other embodiments are in the claims.

What is claimed is:
 1. A P-stereogenic group of formula (IA), (IB),(IC), or (ID):

wherein

is a single carbon-carbon bond or a double carbon-carbon bond; each ofR¹ and R² is independently an optionally substituted C₁₋₆ alkyl oroptionally substituted C₆₋₁₀ aryl, or R¹ and R², together with the atomsto which each is attached, combine to form an optionally substituted 5-to 8-membered ring; and each of R³ and R⁴ is independently H, optionallysubstituted C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.
 2. TheP-stereogenic group of claim 1, wherein the P-stereogenic group is offormula (IA) or (IB).
 3. The P-stereogenic group of claim 1 or 2,wherein R¹ and R², together with the atoms to which each is attached,combine to form an optionally substituted 5- to 8-membered ring.
 4. TheP-stereogenic group of claim 3, wherein the optionally substituted 5- to8-membered ring is an optionally substituted 5- to 8-memberedcarbocyclic ring.
 5. The P-stereogenic group of claim 4, wherein theoptionally substituted 5- to 8-membered ring is an optionallysubstituted 6-membered carbocyclic ring.
 6. The P-stereogenic group ofclaim 1, wherein the P-stereogenic group is of the following structure:


7. The P-stereogenic group of any one of claims 1 to 6, wherein R³ is H.8. The P-stereogenic group of any one of claims 1 to 6, wherein R⁴ is H.9. The P-stereogenic group of any one of claims 1 to 6, wherein R³ andR⁴ are each H.
 10. The P-stereogenic group of any one of claims 1 to 9,wherein the P-stereogenic group is of formula (IIA), (IIB), (IIA′), or(IIB′).
 11. A compound of formula (IIIA), (IIIB), (IIIC), or (IIID):

wherein

is a single carbon-carbon bond or a double carbon-carbon bond; A is anoptionally substituted C₁₋₁₂ alkyl, optionally substituted C₃₋₁₀cycloalkyl, optionally substituted C₃₋₁₀ cycloalkyl-C₁₋₆-alkyl,optionally substituted C₁₋₉ heterocyclyl, optionally substituted C₁₋₉heterocyclyl-C₁₋₆-alkyl, sugar analogue, nucleoside, nucleotide, oroligonucleotide; each of R¹ and R² is independently an optionallysubstituted C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl, or R¹ andR², together with the atoms to which each is attached, combine to forman optionally substituted 5- to 8-membered ring; and each of R³ and R⁴is independently H, optionally substituted C₁₋₆ alkyl, or optionallysubstituted C₆₋₁₀ aryl.
 12. The compound of claim 11, wherein thecompound is of formula (IIIA) or (IIIB).
 13. The compound of claim 11 or12, wherein R¹ and R², together with the atoms to which each isattached, combine to form an optionally substituted 5- to 8-memberedring.
 14. The compound of claim 13, wherein the optionally substituted5- to 8-membered ring is an optionally substituted 5- to 8-memberedcarbocyclic ring.
 15. The compound of claim 14, wherein the optionallysubstituted 5- to 8-membered ring is an optionally substituted6-membered carbocyclic ring.
 16. The compound of claim 11, wherein thecompound is of the following structure:


17. The compound of any one of claims 11 to 16, wherein R³ is H.
 18. Thecompound of any one of claims 11 to 16, wherein R⁴ is H.
 19. Thecompound of any one of claims 11 to 16, wherein R³ and R⁴ are each H.20. The compound of any one of claims 11 to 19, wherein the compound isof formula (IVA), (IVB), (IVA′), or (IVB′).
 21. A nucleosidephosphoramidite comprising a sugar bonded to a nucleobase and to aphosphoramidite of the following structure:

wherein

is a single carbon-carbon bond or a double carbon-carbon bond; each ofR¹ and R² is independently an optionally substituted C₁₋₆ alkyl oroptionally substituted C₆₋₁₀ aryl, or R¹ and R², together with the atomsto which each is attached, combine to form an optionally substituted 5-to 8-membered ring; and each of R³ and R⁴ is independently H, optionallysubstituted C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl.
 22. Thenucleoside phosphoramidite of claim 21, wherein the nucleosidephosphoramidite is of formula (IVA) or (IVB).
 23. The nucleosidephosphoramidite of claim 21, wherein the nucleoside phosphoramidite isof the following structure:

wherein B¹ is a nucleobase; Y¹ is H or C₁₋₆ alkyl; R⁵ is H, O-protectedhydroxyl, optionally substituted C₁₋₆ alkoxy, or halogen; and R⁶ is ahydroxyl protecting group.
 24. The nucleoside phosphoramidite of claim23, wherein the nucleoside phosphoramidite is of formula (VIA) or (VIB).25. The nucleoside phosphoramidite of claim 23 or 24, wherein R⁵ ishydrogen, halogen, or optionally substituted C₁₋₆ alkoxy.
 26. Thenucleoside phosphoramidite of claim 23 or 24, wherein R⁵ is hydrogen,fluoro, or methoxy.
 27. The nucleoside phosphoramidite of any one ofclaims 23 to 26, wherein R⁶ is dimethoxytrityl.
 28. The nucleosidephosphoramidite of any one of claims 21 to 27, wherein R¹ and R²,together with the atoms to which each is attached, combine to form anoptionally substituted 5- to 8-membered ring.
 29. The nucleosidephosphoramidite of claim 28, wherein the optionally substituted 5- to8-membered ring is an optionally substituted 5- to 8-memberedcarbocyclic ring.
 30. The nucleoside phosphoramidite of claim 29,wherein the optionally substituted 5- to 8-membered ring is anoptionally substituted 6-membered carbocyclic ring.
 31. The nucleosidephosphoramidite of any one of claims 21 to 30, wherein thephosphoramidite is of the following structure:


32. The nucleoside phosphoramidite of any one of claims 11 to 31,wherein R³ is H.
 33. The nucleoside phosphoramidite of any one of claims11 to 31, wherein R⁴ is H.
 34. The nucleoside phosphoramidite of any oneof claims 11 to 31, wherein R³ and R⁴ are each H.
 35. A compound offormula:

wherein

is a single carbon-carbon bond or a double carbon-carbon bond; X is ahalogen or pseudohalogen; each of R¹ and R² is independently anoptionally substituted C₁₋₆ alkyl or optionally substituted C₆₋₁₀ aryl,or R¹ and R², together with the atoms to which each is attached, combineto form an optionally substituted 5- to 8-membered ring; and each of R³and R⁴ is independently H, optionally substituted C₁₋₆ alkyl, oroptionally substituted C₆₋₁₀ aryl.
 36. The compound of claim 35, whereinthe compound is of formula (VIIIA) or (VIIIB).
 37. The compound of claim35 or 36, wherein R¹ and R², together with the atoms to which each isattached, combine to form an optionally substituted 5- to 8-memberedring.
 38. The compound of claim 37, wherein the optionally substituted5- to 8-membered ring is an optionally substituted 5- to 8-membercarbocyclic ring.
 39. The compound of claim 38, wherein the optionallysubstituted 5- to 8-membered ring is an optionally substituted 6-membercarbocyclic ring.
 40. The compound of claim 35, wherein the compound isof the following structure:


41. The compound of claim 40, wherein the compound is of formula (IXA),(IXB), (IXA′), or (IXB′).
 42. The compound of any one of claims 35 to41, wherein R³ is H.
 43. The compound of any one of claims 35 to 41,wherein R⁴ is H.
 44. The compound of any one of claims 35 to 41, whereinR³ and R⁴ are each H.
 45. A method of preparing a composition comprisingan oligonucleotide comprising a stereochemically enrichedinternucleoside phosphorothioate, the method comprising (i) reacting thenucleoside phosphoramidite of any one of claims 21 to 34 with a couplingactivator and a nucleoside comprising a 5′-hydroxyl or anoligonucleotide comprising a 5′-hydroxyl, (ii) reacting with anelectrophilic source of acyl, and (iii) reacting with a sulfurizingagent to produce the oligonucleotide comprising a stereochemicallyenriched internucleoside phosphorothioate triester.
 46. The method ofclaim 45 further comprising converting the phosphorothioate triesterinto a phosphorothioate diester by reacting the phosphorothioatetriester with an aqueous base.
 47. The method of claim 45 or 46, whereinthe coupling activator is BTT, PhIMT, or CMPT.
 48. The method of claim47, wherein the coupling activator is CMPT.
 49. The method of any one ofclaims 45 to 48, wherein the nucleoside is a 2-deoxyribonucleoside. 50.The method of any one of claims 45 to 49, wherein the electrophilicsource of acyl is an acid anhydride.
 51. The method of claim 50, whereinthe acid anhydride is acetic anhydride or trifluoroacetic anhydride. 52.The method of any one of claims 45 to 51, wherein the sulfurizing agentis 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione(DDTT).
 53. A method of preparing the nucleoside phosphoramiditecomprising a sugar bonded to a nucleobase and phosphoramidite of thefollowing structure:

wherein

is a single carbon-carbon bond or a double carbon-carbon bond; each ofR¹ and R² is independently an optionally substituted C₁₋₆ alkyl oroptionally substituted C₆₋₁₀ aryl, or R¹ and R², together with the atomsto which each is attached, combine to form an optionally substituted 5-to 8-membered ring; and each of R³ and R⁴ is independently H, optionallysubstituted C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl; the methodcomprising reacting a sugar bonded to a nucleobase with a compound offormula (VIIIA), (VIIIB), (VIIIC), or (VIIID):

wherein X is a halogen or pseudohalogen.
 54. The method of claim 53,wherein the nucleoside phosphoramidite is of formula (VA) or (VB), andthe method comprises reacting a sugar bonded to a nucleobase with acompound of formula (VIIIA) or (VIIIB).
 55. An oligonucleotidecomprising one or more internucleoside groups independently selectedfrom the group consisting of linkers of formula (XIA) and (XIB):

where

is a single carbon-carbon bond or a double carbon-carbon bond; each ofR¹ and R² is independently an optionally substituted C₁₋₆ alkyl oroptionally substituted C₆₋₁₀ aryl, or R¹ and R², together with the atomsto which each is attached, combine to form an optionally substituted 5-to 8-membered ring; each of R³ and R⁴ is independently H, optionallysubstituted C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl; and R⁷ isacyl.
 56. An oligonucleotide comprising one or more internucleosidegroups independently selected from the group consisting of linkers offormula (XIIA) and (XIIIB):

where

is a single carbon-carbon bond or a double carbon-carbon bond; each ofR¹ and R² is independently an optionally substituted C₁₋₆ alkyl oroptionally substituted C₆₋₁₀ aryl, or R¹ and R², together with the atomsto which each is attached, combine to form an optionally substituted 5-to 8-membered ring; each of R³ and R⁴ is independently H, optionallysubstituted C₁₋₆ alkyl, or optionally substituted C₆₋₁₀ aryl; and R⁷ isacyl.